Method for amplifying nucleic acid sequence

ABSTRACT

A convenient and effective method for amplifying a nucleic acid sequence characterized by effecting a DNA synthesis reaction in the presence of chimeric oligonucleotide primers; a method for supplying a large amount of DNA amplification fragments; an effective method for amplifying a nucleic acid sequence by combining the above method with another nucleic acid sequence amplification method; a method for detecting a nucleic acid sequence for detecting or quantitating a microorganism such as a virus, a bacterium, a fungus or a yeast; and a method for detecting a DNA amplification fragment obtained by the above method in situ.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is continuation of co-pending application Ser. No. 09/935,338,filed Aug. 23, 2001, which itself is a continuation-in-part applicationof PCT international application No. PCT/JP00/01534 filed Mar. 14, 2000,which designated the United States, the entire contents of which areincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting a target nucleicacid which is useful in a field of clinical medicine and a method forsynthesizing a DNA which is useful in a field of genetic engineering. Itrelates to a method for amplifying a nucleic acid as a template and amethod for detecting a target nucleic acid amplified by said method.

2. Description of Related Art

DNA synthesis is used for various purposes in studies in a field ofgenetic engineering. Most of the DNA synthesis with the exception ofthat of a short-chain DNA such as an oligonucleotide is carried out byenzymatic methods in which a DNA polymerase is utilized. An example ofthe methods is the polymerase chain reaction (PCR) method as describedin U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 in detail. Anotherexample is the reverse transcription-PCR (RT-PCR) method, which is acombination of the PCR and a reverse transcriptase reaction, asdescribed in Trends in Biotechnology, 10:146-152 (1992). The developmentof the above-mentioned methods has enabled the amplification of a regionof interest from a DNA or an RNA.

The above-mentioned DNA synthesis methods are conducted, for example,using a reaction that consists of three steps. The three steps are astep of dissociating (denaturing) a double-stranded DNA intosingle-stranded DNAs, a step of annealing a primer to thesingle-stranded DNA and a step of synthesizing (extending) acomplementary strand from the primer in order to amplify a region of aDNA of interest. Alternatively, they are conducted using a reactiondesignated as “the shuttle PCR” (“PCR no saizensen” (Recent advances inPCR methodology: Basic methodology and it's application), TanpakushitsuKakusan Kouso, Bessatsu, (Protein, Nucleic Acid and Enzyme, Supplement),41(5):425-428 (1996)) in which two of the three steps, that is, the stepof annealing the primer and the step of extending are carried out at thesame temperature.

Alternatively, the ligase chain reaction (LCR) method as described in EP320,308 published on Jun. 14, 1989 or the transcription-basedamplification system (TAS) method as described in PCR Protocols,Academic Press Inc., 1990, pp. 245-252 may be used. The four methods asmentioned above require repeating a reaction at a high temperature andthat at a low temperature several times in order to regenerate asingle-stranded target molecule for the next amplification cycle. Thereaction system should be conducted using discontinuous phases or cyclesbecause the reaction is restricted by temperature as described above.

Thus, the methods require the use of an expensive thermal cycler thatcan strictly adjust a wide range of temperature over time. Furthermore,the reaction requires time for adjusting the temperature to the two orthree predetermined ones. The loss of time increases in proportion tothe cycle number.

Nucleic acid amplification methods that can be carried out isothermallyhave been developed in order to solve the problems. Examples thereofinclude the strand displacement amplification (SDA) method as describedin JP-B 7-114718, the self-sustained sequence replication (3SR) method,the nucleic acid sequence based amplification (NASBA) method asdescribed in Japanese Patent No. 2650159, the transcription-mediatedamplification (TMA) method, the Qβ replicase method as described inJapanese Patent No. 2710159 and the various modified SDA methods asdescribed in U.S. Pat. No. 5,824,517, WO 99/09211, WO 95/25180 and WO99/49081. A method of isothermal enzymatic synthesis of anoligonucleotide is described in U.S. Pat. No. 5,916,777. Extension froma primer and/or annealing of a primer to a single-stranded extensionproduct (or to an original target sequence) followed by extension fromthe primer take place in parallel in a reaction mixture incubated at aconstant temperature in the reaction of these methods of isothermalnucleic acid amplification or synthesis of an oligonucleotide.

Among the isothermal nucleic acid amplification methods, the SDA methodis an example of systems in which a DNA is finally amplified. The SDAmethod is a method for amplifying a target nucleic acid sequence (and acomplementary strand thereof) in a sample by displacement of doublestrands using a DNA polymerase and a restriction endonuclease. Themethod requires four primers used for the amplification, two of whichshould be designed to contain a recognition site for the restrictionendonuclease. The method requires the use of a modifieddeoxyribonucleotide triphosphate as a substrate for DNA synthesis inlarge quantities. An example of the modified deoxyribonucleotidetriphosphates is an (α-S) deoxyribonucleotide triphosphate in which theoxygen atom of the phosphate group at the α-position is replaced by asulfur atom (S). The problem of running cost associated with the use ofthe modified deoxyribonucleotide triphosphate becomes serious if thereaction is routinely conducted, for example, for genetic test.Furthermore, the incorporation of the modified nucleotide such as the(α-S) deoxyribonucleotide into the amplified DNA fragment in the methodmay abolish the cleavability of the amplified DNA fragment with arestriction enzyme, for example, when it is subjected to a restrictionenzyme fragment length polymorphism (RFLP) analysis.

The modified SDA method as described in U.S. Pat. No. 5,824,517 is a DNAamplification method that uses a chimeric primer that is composed of anRNA and a DNA and has as an essential element a structure in which DNAis positioned at least at the 3′-terminus. The modified SDA method asdescribed in WO 99/09211 requires the use of a restriction enzyme thatgenerates a 3′-protruding end. The modified SDA method as described inWO 95/25180 requires the use of at least two pairs of primers. Themodified SDA method as described in WO 99/49081 requires the use of atleast two pairs of primers and at least one modified deoxyribonucleotidetriphosphate. On the other hand, the method for synthesizing anoligonucleotide as described in U.S. Pat. No. 5,916,777 comprisessynthesizing a DNA using a primer having a ribonucleotide at the3′-terminus, completing a reaction using the primer, introducing a nickbetween the primer and an extended strand in an primer-extended strandwith an endonuclease to separate them, digesting a template andrecovering the primer to reuse it. It is required to isolate the primerfrom the reaction system and then anneal it to the template again inorder to reuse the primer in the method. Additionally, the LAMP(Loop-mediated Isothermal Amplification) method as described in WO00/28082 requires four primers for amplification and the productsamplified using the method are DNAs having varying size in which thetarget regions for the amplification are repeated.

As described above, the conventional isothermal nucleic acidamplification methods still have various problems. Thus, a method foramplifying a nucleic acid at low running cost by which a DNA fragmentthat can be further genetically engineered is obtained has been desired.

The main object of the present invention is to provide a method foramplifying a target nucleic acid which specifically amplifies with highsensitivity a target nucleic acid in a sample by DNA synthesis reactionusing a chimeric oligonucleotide primer, a method for detecting aamplified fragment obtained by said method, a method for producing atarget nucleic acid using said amplification method and a chimericoligonucleotide primer used for these methods.

SUMMARY OF THE INVENTION

As a result of intensive studies, the present inventors have constructedan excellent system for gene amplification reaction. The constructionwas accomplished by developing a method in which a region of a DNA ofinterest is amplified in the presence of a chimeric oligonucleotideprimer having a ribonucleotide positioned at the 3′-terminus or on the3′-terminal side, an endonuclease and a DNA polymerase. Thus, thepresent invention has been completed. The method is a method foramplifying a nucleic acid in which a chimeric oligonucleotide primer isused and herein referred to as ICAN (Isothermal and Chimericprimer-initiated Amplification of Nucleic acids) method.

The first invention of the present invention relates to a method foramplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer for cleavage with an        endonuclease;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with the endonuclease at a        site that contains the ribonucleotide; and    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement.

The second invention of the present invention relates to a method foramplifying a nucleotide sequence using at least two primers,characterized in that the method comprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer for cleavage with an        endonuclease;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with the endonuclease at a        site that contains the ribonucleotide;    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (b);    -   (d) treating a released displaced strand obtained in step (c) as        a template with at least one primer that is different from that        used in step (a) and a DNA polymerase to synthesize a        primer-extended strand that is complementary to the displaced        strand, wherein the primer that is different from that used in        step (a) is a chimeric oligonucleotide primer that is        substantially complementary to the nucleotide sequence of the        displaced strand and contains a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer for        cleavage with an endonuclease;    -   (e) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (d) with the endonuclease at a        site that contains the ribonucleotide; and    -   (f) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (e) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (e).

The method of the first and second inventions of the present inventionmay be conducted isothermally. The nucleotide sequence as the templatemay be a DNA sequence. A step of preparing a single-stranded cDNA by areverse transcription reaction using reverse transcriptase and an RNA asa template may be comprised prior to step (a) of the first and secondinventions. The single-stranded cDNA may be used as the nucleotidesequence as the template. Both of a single-stranded DNA and adouble-stranded DNA can be preferably used as the DNA as the template inthe first and second inventions of the present inventions. If adouble-stranded DNA is used as the template, the method of the presentinvention may be conducted after a pretreatment step of denaturing thedouble-stranded DNA into single-stranded DNAs.

In the above-mentioned inventions, the extension from the primer isconducted using a DNA polymerase having a strand displacement activity.A DNA polymerase selected from the group consisting of Klenow fragmentof DNA polymerase I from Escherichia coli, Bst DNA polymerase lacking5′3′ exonuclease from Bacillus stearothermophilus and Bca DNA polymeraselacking 5′3′ exonuclease from Bacillus caldotenax can be preferably usedin the present invention. Additionally, an endoribonuclease can bepreferably used as the endonuclease. The endoribonuclease that can beused include, but are not limited to, RNase H, for example.

The third invention of the present invention relates to a method foramplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) preparing a reaction mixture by mixing a nucleic acid as a        template, a deoxyribonucleotide triphosphate, a DNA polymerase        having a strand displacement activity, at least one primer and        an endonuclease that cleaves an extended strand generated from        the primer, wherein the primer is a chimeric oligonucleotide        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid as the template and contains a        deoxyribonucleotide and a ribonucleotide, the ribonucleotide        being positioned at the 3′-terminus or on the 3′-terminal side        of the primer for cleavage with the endonuclease; and    -   (b) incubating the reaction mixture for a sufficient time to        generate a reaction product.

Examples of the nucleotide sequences as the templates that can be usedin the third invention include a nucleotide sequence selected from thegroup consisting of a single-stranded DNA, a double-stranded DNAdenatured into single-stranded DNAs and a cDNA obtained by a reversetranscription reaction from an RNA. Two or more chimeric oligonucleotideprimers may be contained in the reaction mixture. The DNA polymerasehaving a strand displacement activity and the endonuclease used in thefirst and second inventions can be preferably used in this invention.

The primer used in the first to third inventions of the presentinvention is a chimeric oligonucleotide primer. For example, a chimericoligonucleotide having a structure in which at least one, preferably twoor more successive ribonucleotide residues are attached at the3′-terminus or on the 3′-terminal side of the primer can be used.

The template used in the first to third inventions of the presentinvention may be a nucleic acid that is amplified beforehand by anucleic acid amplification method. For example, the TAS method, the 3SRmethod, the NASBA method, the TMA method, the Qβ replicase method, thePCR method, the LCR method and the SDA method can be utilized as thenucleic acid amplification method although any methods for amplifying anucleic acid can be used without limitation. The method of the presentinvention can be used in combination with these nucleic acidamplification methods.

A random primer or a degenerate primer can be used in the nucleic acidamplification reaction. For example, without limitation, a primer havinga random sequence or a degenerate sequence at least at the 3′-terminusor on the 3′-terminal side can be preferably used.

The fourth invention of the present invention relates to a chimericoligonucleotide primer that can be used for the first to thirdinventions. The primer is characterized in that it contains adeoxyribonucleotide and a ribonucleotide and has a structure in whichthe ribonucleotide is position at the 3′-terminus or on the 3′-terminalside of the primer. For example, a chimeric oligonucleotide primer thatcontains at least one, preferably two or more successive ribonucleotideresidues and enables the extension of a DNA strand from the 3′-terminusthereof may be used. Such a primer is designed such that it is cleavedby the action of a ribonuclease such as RNase H at the 3′-terminus ofthe ribonucleotide residue.

The fifth invention of the present invention relates to a DNA polymerasehaving a strand displacement activity, an endonuclease, and a kit thatcontains them used for the first to third inventions.

The sixth invention of the present invention relates to a method fordetecting a target nucleic acid, characterized in that the methodcomprises amplifying a target nucleic acid by the method for amplifyinga nucleotide sequence of the first to third inventions of the presentinvention and then detecting the nucleic acid. The methods of detectioninclude a method in which the target nucleic acid is detected using aribonucleotide (RNA) probe labeled with two or more fluorescent dyespositioned at a distance that results in a quenching state.

The seventh invention of the present invention relates to a DNApolymerase having a strand displacement activity, an endonuclease and akit that contains them used for the method for detecting a targetnucleic acid of the sixth invention of the present invention.

The eighth invention of the present invention relates to a method forproducing a material having an immobilized nucleic acid in which thenucleic acid is arrayed in a predefined region, characterized in thatthe method comprises arraying and immobilizing the nucleic acidamplified by the method for amplifying a nucleotide sequence of thefirst to third inventions of the present invention in a predefinedregion on a substrate. A method in which a single-stranded nucleic acidsubstantially free of a complementary strand thereto is amplified, andarrayed and immobilized in the predefined region is particularlypreferable.

The ninth invention of the present invention relates to a materialhaving an immobilized nucleic acid in which the nucleic acid is arrayedin a predefined region produced by the method of the eighth invention ofthe present invention. A material having an immobilized nucleic acid inwhich a single-stranded nucleic acid substantially free of acomplementary strand thereto is arrayed and immobilized in thepredefined region is particularly preferable.

The tenth invention of the present invention relates to a method fordetecting a target nucleic acid in a sample, characterized in that themethod uses the material having an immobilized nucleic acid in which thenucleic acid is arrayed in a predefined region of the ninth invention ofthe present invention to detect a nucleic acid that hybridizes with thenucleic acid arrayed and immobilized in the predefined region on thematerial.

The eleventh invention of the present invention relates to a method forproducing a nucleic acid in large quantities, characterized in that themethod comprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer for cleavage with an        endonuclease;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with the endonuclease at a        site that contains the ribonucleotide; and    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement.

The twelfth invention of the present invention relates to a method forproducing a nucleic acid in large quantities using at least two primers,characterized in that the method comprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer for cleavage with an        endonuclease;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with the endonuclease at a        site that contains the ribonucleotide;    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (b);    -   (d) treating a released displaced strand obtained in step (c) as        a template with at least one primer that is different from that        used in step (a) and a DNA polymerase to synthesize a        primer-extended strand that is complementary to the displaced        strand, wherein the primer that is different from that used in        step (a) is a chimeric oligonucleotide primer that is        substantially complementary to the nucleotide sequence of the        displaced strand and contains a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer for        cleavage with an endonuclease;    -   (e) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (d) with the endonuclease at a        site that contains the ribonucleotide; and    -   (f) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (e) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (e).

The thirteenth invention of the present invention relates to a methodfor producing a nucleic acid in large quantities, characterized in thatthe method comprises:

-   -   (a) preparing a reaction mixture by mixing a nucleic acid as a        template, a deoxyribonucleotide triphosphate, a DNA polymerase        having a strand displacement activity, at least one primer and        an endonuclease that cleaves an extended strand generated from        the primer, wherein the primer is a chimeric oligonucleotide        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid as the template and contains a        deoxyribonucleotide and a ribonucleotide, the ribonucleotide        being positioned at the 3′-terminus or on the 3′-terminal side        of the primer for cleavage with the endonuclease; and    -   (b) incubating the reaction mixture for a sufficient time to        generate a reaction product.

The fourteenth invention of the present invention relates to a methodfor amplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) amplifying a nucleic acid containing a sequence to be        amplified by a nucleic acid amplification reaction to prepare a        nucleic acid as a template;    -   (b) treating the nucleic acid as the template obtained in        step (a) with at least one primer that is substantially        complementary to the nucleotide sequence of the nucleic acid and        a DNA polymerase to synthesize a primer-extended strand that is        complementary to the template, wherein the primer is a chimeric        oligonucleotide primer containing a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer for        cleavage with an endonuclease;    -   (c) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (b) with the endonuclease at a        site that contains the ribonucleotide; and    -   (d) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (c) to effect a strand displacement.

The fifteenth invention of the present invention relates to a method foramplifying a nucleotide sequence using at least two primers,characterized in that the method comprises:

-   -   (a) amplifying a nucleic acid containing a sequence to be        amplified by a nucleic acid amplification reaction to prepare a        nucleic acid as a template;    -   (b) treating the nucleic acid as the template obtained in        step (a) with at least one primer that is substantially        complementary to the nucleotide sequence of the nucleic acid and        a DNA polymerase to synthesize a primer-extended strand that is        complementary to the template, wherein the primer is a chimeric        oligonucleotide primer containing a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer for        cleavage with an endonuclease;    -   (c) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (b) with the endonuclease at a        site that contains the ribonucleotide;    -   (d) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (c) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (c);    -   (e) treating a released displaced strand obtained in step (d) as        a template with at least one primer that is different from that        used in step (b) and a DNA polymerase to synthesize a        primer-extended strand that is complementary to the displaced        strand, wherein the primer that is different from that used in        step (b) is a chimeric oligonucleotide primer that is        substantially complementary to the nucleotide sequence of the        displaced strand and contains a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer for        cleavage with an endonuclease;    -   (f) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (e) with the endonuclease at a        site that contains the ribonucleotide; and    -   (g) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (f) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (f).

The sixteenth invention of the present invention relates to a method foramplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) amplifying a nucleic acid containing a sequence to be        amplified by a nucleic acid amplification reaction to prepare a        nucleic acid as a template;    -   (b) preparing a reaction mixture by mixing the nucleic acid as        the template obtained in step (a), a deoxyribonucleotide        triphosphate, a DNA polymerase having a strand displacement        activity, at least one primer and an endonuclease that cleaves        an extended strand generated from the primer, wherein the primer        is a chimeric oligonucleotide primer that is substantially        complementary to the nucleotide sequence of the nucleic acid as        the template and contains a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer for        cleavage with the endonuclease; and    -   (c) incubating the reaction mixture for a sufficient time to        generate a reaction product.

In the fourteenth to sixteenth inventions of the present invention, anucleic acid containing a sequence to be amplified is amplifiedbeforehand by a nucleic acid amplification reaction. The amplificationproduct is then used as a nucleic acid as a template in the method ofthe first to third inventions of the present invention. For example, theTAS method, the 3SR method, the NASBA method, the TMA method, the Qβreplicase method, the PCR method, the LCR method and the SDA method canbe used as the nucleic acid amplification method used in the fourteenthto sixteenth inventions although any methods for amplifying a nucleicacid can be used without limitation.

A random primer or a degenerate primer can be used in the nucleic acidamplification reaction. For example, without limitation, a primer havinga random sequence or a degenerate sequence at least at the 3′-terminusor on the 3′-terminal side can be preferably used.

The seventeenth invention of the present invention relates to a methodfor amplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with an endonuclease at a site        that contains the ribonucleotide; and    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement.

The eighteenth invention of the present invention relates to a methodfor amplifying a nucleotide sequence using at least two primers,characterized in that the method comprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with an endonuclease at a site        that contains the ribonucleotide;    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (b);    -   (d) treating a released displaced strand obtained in step (c) as        a template with at least one primer that is different from that        used in step (a) and a DNA polymerase to synthesize a        primer-extended strand that is complementary to the displaced        strand, wherein the primer that is different from that used in        step (a) is a chimeric oligonucleotide primer that is        substantially complementary to the nucleotide sequence of the        displaced strand and contains a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer;    -   (e) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (d) with an endonuclease at a site        that contains the ribonucleotide; and    -   (f) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (e) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (e).

The nineteenth invention of the present invention relates to a methodfor amplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) preparing a reaction mixture by mixing a nucleic acid as a        template, a deoxyribonucleotide triphosphate, a DNA polymerase        having a strand displacement activity, at least one primer and        an endonuclease that cleaves an extended strand generated from        the primer, wherein the primer is a chimeric oligonucleotide        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid as the template and contains a        deoxyribonucleotide and a ribonucleotide, the ribonucleotide        being positioned at the 3′-terminus or on the 3′-terminal side        of the primer; and    -   (b) incubating the reaction mixture for a sufficient time to        generate a reaction product.

The twentieth invention of the present invention relates to a method foramplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer, wherein an endonuclease cleaves        at a site that contains the ribonucleotide;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with the endonuclease at the        site that contains the ribonucleotide; and    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement.

The twenty-first invention of the present invention relates to a methodfor amplifying a nucleotide sequence using at least two primers,characterized in that the method comprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a deoxyribonucleotide and a ribonucleotide, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer, wherein an endonuclease cleaves        at a site that contains the ribonucleotide;    -   (b) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (a) with the endonuclease at the        site that contains the ribonucleotide;    -   (c) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (b) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (b);    -   (d) treating a released displaced strand obtained in step (c) as        a template with at least one primer that is different from that        used in step (a) and a DNA polymerase to synthesize a        primer-extended strand that is complementary to the displaced        strand, wherein the primer that is different from that used in        step (a) is a chimeric oligonucleotide primer that is        substantially complementary to the nucleotide sequence of the        displaced strand and contains a deoxyribonucleotide and a        ribonucleotide, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer, wherein an        endonuclease cleaves at a site that contains the ribonucleotide;    -   (e) cleaving the primer-extended strand of a double-stranded        nucleic acid obtained in step (d) with the endonuclease at the        site that contains the ribonucleotide; and    -   (f) extending a nucleotide sequence that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (e) to effect a strand displacement,        wherein a double-stranded nucleic acid containing a regenerated        primer-extended strand is reused in step (e).

The twenty-second invention of the present invention relates to a methodfor amplifying a nucleotide sequence, characterized in that the methodcomprises:

-   -   (a) preparing a reaction mixture by mixing a nucleic acid as a        template, a deoxyribonucleotide triphosphate, a DNA polymerase        having a strand displacement activity, at least one primer and        an endonuclease that cleaves an extended strand generated from        the primer, wherein the primer is a chimeric oligonucleotide        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid as the template and contains a        deoxyribonucleotide and a ribonucleotide, the ribonucleotide        being positioned at the 3′-terminus or on the 3′-terminal side        of the primer, wherein the endonuclease cleaves at a site that        contains the ribonucleotide; and    -   (b) incubating the reaction mixture for a sufficient time to        generate a reaction product.

The twenty-third invention of the present invention relates to a methodfor determining a nucleotide sequence of a nucleic acid, characterizedin that the method comprises amplifying a nucleotide sequence accordingto the method of any one of the first to third and fourteenth totwenty-second inventions.

The twenty-fourth invention of the present invention relates to a methodfor amplifying a target nucleic acid, characterized in that the methodcomprises:

-   -   (a) preparing a reaction mixture by mixing a nucleic acid as a        template, a deoxyribonucleotide triphosphate, a DNA polymerase        having a strand displacement activity, at least one primer and        an RNase H, wherein the primer is a chimeric oligonucleotide        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid as the template and contains a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer; and    -   (b) incubating the reaction mixture for a sufficient time to        generate a reaction product.

In the twenty-fourth invention of the present invention, a reactionmixture that further contains a chimeric oligonucleotide primer having asequence that is substantially homologous to the nucleotide sequence ofthe nucleic acid as the template can be used.

The twenty-fifth invention of the present invention relates to a methodfor amplifying a nucleic acid, characterized in that the methodcomprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template        and synthesize a double-stranded nucleic acid, wherein the        primer is a chimeric oligonucleotide primer containing a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) extending a nucleic acid that is complementary to the        double-stranded nucleic acid as a template obtained in the        previous step using a DNA polymerase having a strand        displacement activity in the presence of an RNase H to effect a        strand displacement and synthesize a displaced strand and a        double-stranded nucleic acid; and    -   (c) reusing in step (b) the double-stranded nucleic acid        obtained in step (b) as a template.

The twenty-sixth invention of the present invention relates to a methodfor amplifying a nucleic acid using at least two primers, characterizedin that the method comprises:

-   -   (a) treating a nucleic acid as a template with at least one        primer that is substantially complementary to the nucleotide        sequence of the nucleic acid and a DNA polymerase to synthesize        a primer-extended strand that is complementary to the template,        wherein the primer is a chimeric oligonucleotide primer        containing a ribonucleotide as well as at least one selected        from the group consisitng of a deoxyribonucleotide and a        nucleotide analog, the ribonucleotide being positioned at the        3′-terminus or on the 3′-terminal side of the primer;    -   (b) extending a nucleic acid that is complementary to the        double-stranded nucleic acid as a template obtained in the        previous step using a DNA polymerase having a strand        displacement activity in the presence of an RNase H to effect a        strand displacement and synthesize a displaced strand and a        double-stranded nucleic acid;    -   (c) reusing in step (b) the double-stranded nucleic acid        obtained in step (b) as a template;    -   (d) treating a displaced strand obtained in step (b) as a        template with at least one primer that is different from that        used in step (a) and a DNA polymerase to synthesize a        primer-extended strand that is complementary to the displaced        strand, wherein the primer that is different from that used in        step (a) is a chimeric oligonucleotide primer that is        substantially complementary to the nucleotide sequence of the        displaced strand and contains a ribonucleotide as well as at        least one selected from the group consisitng of a        deoxyribonucleotide and a nucleotide analog, the ribonucleotide        being positioned at the 3′-terminus or on the 3′-terminal side        of the primer;    -   (e) extending a nucleic acid that is complementary to the        double-stranded nucleic acid as a template obtained in the        previous step using a DNA polymerase having a strand        displacement activity in the presence of an RNase H to effect a        strand displacement and synthesize a displaced strand and a        double-stranded nucleic acid; and    -   (f) reusing in step (e) the double-stranded nucleic acid        obtained in step (e) as a template.

In the twenty-fifth or twenty-sixth invention of the present invention,a DNA polymerase having a strand displacement activity can be used asthe DNA polymerase.

The twenty-seventh invention of the present invention relates to methodfor amplifying a nucleic acid, characterized in that the methodcomprises:

-   -   (a) treating a double-stranded nucleic acid as a template with        two primers that are substantially complementary to the        nucleotide sequences of the respective strands of the        double-stranded nucleic acid and a DNA polymerase having a        strand displacement activity to synthesize primer-extended        strands that are complementary to the template and obtain a        double-stranded nucleic acid consisting of the synthesized        primer-extended strands being annealed each other, wherein each        primer is a chimeric oligonucleotide primer containing a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) cleaving the double-stranded nucleic acid consisting of the        primer-extended strands obtained in step (a) with the        endonuclease at sites that contain the ribonucleotides; and    -   (c) extending nucleic acids that are complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-termini of the respective primer portions        of the double-stranded nucleic acid in which the primer-extended        strands are cleaved obtained in step (b) to effect strand        displacements and obtain a double-stranded nucleic acid        consisting of the template and the primer-extended strand.

The twenty-eighth invention of the present invention relates to a methodfor amplifying a nucleic acid, characterized in that the methodcomprises:

-   -   (a) treating a double-stranded nucleic acid as a template with        two primers that are substantially complementary to the        nucleotide sequences of the respective strands of the        double-stranded nucleic acid and a DNA polymerase having a        strand displacement activity to synthesize primer-extended        strands that are complementary to the template and obtain a        double-stranded nucleic acid consisting of the synthesized        primer-extended strands being annealed each other, wherein each        primer is a chimeric oligonucleotide primer containing a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) cleaving the double-stranded nucleic acid consisting of the        primer-extended strands obtained in step (a) with the        endonuclease at sites that contain the ribonucleotides; and    -   (c) extending nucleic acids that are complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-termini of the respective primer portions        of the double-stranded nucleic acid in which the primer-extended        strands are cleaved obtained in step (b) to effect strand        displacements and obtain a double-stranded nucleic acid        consisting of the primer-extended strands being annealed each        other.

The twenty-ninth invention of the present invention relates to a methodfor amplifying a nucleic acid, characterized in that the methodcomprises:

-   -   (a) treating a double-stranded nucleic acid as a template with        two primers that are substantially complementary to the        nucleotide sequences of the respective strands of the        double-stranded nucleic acid and a DNA polymerase having a        strand displacement activity to synthesize primer-extended        strands that are complementary to the template and obtain a        double-stranded nucleic acid consisting of the synthesized        primer-extended strands being annealed each other, wherein each        primer is a chimeric oligonucleotide primer containing a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) cleaving the double-stranded nucleic acid consisting of the        primer-extended strands obtained in step (a) with the        endonuclease at sites that contain the ribonucleotides;    -   (c) extending nucleic acids that are complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-termini of the respective primer portions        of the double-stranded nucleic acid in which the primer-extended        strands are cleaved obtained in step (b) to effect strand        displacements and obtain a double-stranded nucleic acid        consisting of the primer-extended strands being annealed each        other and a double-stranded nucleic acid consisting of the        templates being annealed each other to which the two primers in        step (a) are annealed;    -   (d) extending nucleic acids that are complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-termini of the respective primer portions        of the double-stranded nucleic acid to which the two primers are        annealed obtained in step (c) to effect strand displacements and        obtain a double-stranded nucleic acid consisting of the        primer-extended strands being annealed each other and a        double-stranded nucleic acid consisting of the templates being        annealed each other to which the two primers in step (a) are        annealed; and    -   (e) reusing in step (d) the double-stranded nucleic acid to        which the two primers are annealed obtained in step (d).

The thirtieth invention of the present invention relates to a method foramplifying a nucleic acid, characterized in that the method comprises:

-   -   (a) treating a double-stranded nucleic acid as a template with        two primers that are substantially complementary to the        nucleotide sequences of the respective strands of the        double-stranded nucleic acid and a DNA polymerase having a        strand displacement activity to synthesize primer-extended        strands that are complementary to the template and obtain a        double-stranded nucleic acid consisting of the synthesized        primer-extended strands being annealed each other, wherein each        primer is a chimeric oligonucleotide primer containing a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) cleaving the double-stranded nucleic acid consisting of the        primer-extended strands obtained in step (a) with the        endonuclease at sites that contain the ribonucleotides;    -   (c) extending nucleic acids that are complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-termini of the respective primer portions        of the double-stranded nucleic acid in which the primer-extended        strands are cleaved obtained in step (b) to effect strand        displacements and obtain a double-stranded nucleic acid        consisting of the primer-extended strands being annealed each        other and a double-stranded nucleic acid consisting of the        templates being annealed each other to which the two primers in        step (a) are annealed;    -   (d) extending nucleic acids that are complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-termini of the respective primer portions        of the double-stranded nucleic acid to which the two primers are        annealed obtained in step (c) to effect a strand displacement        and obtain a double-stranded nucleic acid consisting of the        template and the primer-extended strand;    -   (e) cleaving the double-stranded nucleic acid consisting of the        template and the primer-extended strand obtained in step (d)        with the endonuclease at a site that contains the        ribonucleotide; and    -   (f) extending a nucleic acid that is complementary to the        template using a DNA polymerase having a strand displacement        activity from the 3′-terminus of the primer portion of the        double-stranded nucleic acid in which the primer-extended strand        is cleaved obtained in step (e) to synthesize a displaced        strand.

In the twenty-seventh to thirtieth invention, an endoribonuclease suchas an RNase H can be used as the endonuclease.

In the twenty-fourth to thirtieth invention wherein an RNase H is used,an RNase H from Escherichia coli, a bacterium of genus Thermotoga, abacterium of genus Thermus, a bacterium of genus Pyrococcus, a bacteriumof genus Archaeoglobus, a bacterium of genus Bacillus or the like can beused.

In the twenty-fourth to thirtieth invention, an example of the suitablelength of the region to be specifically amplified in the nucleotidesequence of the target nucleic acid is 200 bp or shorter.

For the twenty-fourth to thirtieth invention of the present invention, achimeric oligonucleotide primer represented by general formula below canbe used:5′-dNa-Nb-dNc-3′  General formula(a: an integer of 11 or more; b: an integer of 1 or more; c: 0 or aninteger of 1 or more; dN: deoxyribonucleotide and/or nucleotide analog;N: unmodified ribonucleotide and/or modified ribonucleotide, whereinsome of dNs in dNa may be replaced by Ns, and the nucleotide at the3′-terminus may be modified such that extension from the 3′-terminus bythe action of the DNA polymerase does not take place).

Such chimeric oligonucleotide primers are exemplified by a primer inwhich c is 0 and a primer in which the nucleotide analog isdeoxyriboinosine nucleotide or deoxyribouracil nucleotide, and themodified ribonucleotide is (α-S) ribonucleotide. Furthermore, when sucha chimeric oligonucleotide primer is used, the DNA extension reaction isconducted at a DNA extension reaction temperature suitable for theprimer.

The amplification method of the twenty-fourth to thirtieth invention cancomprise annealing the nucleic acid as the template to the chimericoligonucleotide primer that is substantially complementary to thenucleotide sequence of the nucleic acid in an annealing solutioncontaining a substance that enhances the annealing of the nucleic acidto the primer. For example, the annealing solution may containspermidine and/or propylenediamine. The annealing can be conducted byincubating the annealing solution containing the nucleic acid as thetemplate and the chimeric oligonucleotide primer that is substantiallycomplementary to the nucleotide sequence of the nucleic acid at 90 C orabove and then cooling the solution to a temperature at which theamplification reaction is conducted or below.

The amplification reaction of the twenty-fourth to thirtieth inventioncan be conducted in a buffer containing a buffering component selectedfrom the group consisting of Bicine and HEPES.

In the twenty-fourth to thirtieth invention, for example, a DNApolymerase selected from the group consisting of Klenow fragment of DNApolymerase I from Escherichia coli, Bst DNA polymerase lacking 5′3′exonuclease from Bacillus stearothermophilus and Bca DNA polymeraselacking 5′3′ exonuclease from Bacillus caldotenax can be used as the DNApolymerase having a strand displacement activity.

In one aspect of the twenty-fourth to thirtieth invention, Bca DNApolymerase lacking 5′3′ exonuclease from Bacillus caldotenax is used asthe DNA polymerase having a strand displacement activity and an RNase Hfrom Escherichia coli, a bacterium of genus Pyrococcus or a bacterium ofgenus Archaeoglobus is used as the endonuclease. The RNase H isexemplified by type I RNase H from Escherichia coli, or type II RNase Hfrom a bacterium of genus Pyrococcus or a bacterium of genusArchaeoglobus.

In the twenty-fourth to thirtieth invention, a DNA polymerase having anendonuclease activity can be used. Bca DNA polymerase lacking 5′3′exonuclease from Bacillus caldotenax can be used as such a DNApolymerase and the amplification reaction can be conducted in thepresence of a substance that allows the endonuclease activity of the DNApolymerase to express. The substance that allows the endonucleaseactivity of the Bca DNA polymerase to express is exemplified by amanganese ion.

The method for amplifying a target nucleic acid of the twenty-fourth tothirtieth invention can be conducted in the presence of a substance thatinhibits the reverse transcription activity of the DNA polymerase. Thesubstance that inhibits the reverse transcription activity of the DNApolymerase is exemplified by phosphonoformic acid.

The twenty-fourth to thirtieth invention of the present invention can beconducted using a single-stranded DNA or a double-stranded DNA as thetemplate. If the nucleic acid as the template is a double-stranded DNA,the amplification reaction can be conducted after converting it intosingle-stranded DNAs.

The nucleic acid as the template may be a cDNA obtained by a reversetranscription reaction using an RNA as a template. In one aspect, theamplification reaction is conducted after synthesizing a cDNA by areverse transcription reaction using an RNA as a template. A DNApolymerase having a reverse transcriptase activity can be used for thereverse transcription reaction. For example, the reverse transcriptionreaction and the synthesis of the extended strand that is complementaryto the template can be conducted using one DNA polymerase having areverse transcriptase activity and a strand displacement activity. Sucha DNA polymerase is exemplified by Bst DNA polymerase lacking 5′3′exonuclease from Bacillus stearothermophilus or Bca DNA polymeraselacking 5′3′ exonuclease from Bacillus caldotenax.

In the twenty-fourth to thirtieth invention, the amplification reactioncan be conducted under isothermal conditions. In addition, it can beconducted in the presence of a deoxynucleotide triphosphate analog suchas deoxyuridine triphosphate or a derivative thereof.

The thirty-first invention of the present invention relates to acomposition for amplifying a nucleic acid which contains:

-   -   (a) at least one primer that is substantially complementary to a        nucleotide sequence of a nucleic acid as a template, wherein the        primer is a chimeric oligonucleotide primer that contains a        ribonucleotide as well as at least one selected from the group        consisitng of a deoxyribonucleotide and a nucleotide analog, the        ribonucleotide being positioned at the 3′-terminus or on the        3′-terminal side of the primer;    -   (b) an endonuclease; and    -   (c) a DNA polymerase having a strand displacement activity.

The thirty-second invention of the present invention relates to acomposition for amplifying a nucleic acid which contains:

-   -   (a) at least two primers that are substantially complementary to        nucleotide sequences of respective strands of a double-stranded        nucleic acid as a template, wherein each primer is a chimeric        oligonucleotide primer that contains a ribonucleotide as well as        at least one selected from the group consisitng of a        deoxyribonucleotide and a nucleotide analog, the ribonucleotide        being positioned at the 3′-terminus or on the 3′-terminal side        of the primer;    -   (b) an endonuclease; and    -   (c) a DNA polymerase having a strand displacement activity.

The thirty-third invention of the present invention relates to acomposition for amplifying a nucleic acid obtained by mixing a nucleicacid as a template, a deoxyribonucleotide triphosphate, a DNA polymerasehaving a strand displacement activity, at least one primer and anendonuclease, wherein the primer is a chimeric oligonucleotide primerthat is substantially complementary to the nucleotide sequence of thenucleic acid as the template and contains a ribonucleotide as well as atleast one selected from the group consisitng of a deoxyribonucleotideand a nucleotide analog, the ribonucleotide being positioned at the3′-terminus or on the 3′-terminal side of the primer.

The thirty-fourth invention of the present invention relates tocomposition for amplifying a nucleic acid obtained by mixing a nucleicacid as a template, a deoxyribonucleotide triphosphate, a DNA polymerasehaving a strand displacement activity, at least two primers and anendonuclease, wherein each primer is a chimeric oligonucleotide primerthat is substantially complementary to the nucleotide sequence of eachstrand of the double-stranded nucleic acid as the template and containsa ribonucleotide as well as at least one selected from the groupconsisitng of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer.

The primer contained in the composition of the thirty-first tothirty-fourth invention of the present invention is exemplified by achimeric oligonucleotide primer represented by general formula below:5′-dNa-Nb-dNc-3′  General formula(a: an integer of 11 or more; b: an integer of 1 or more; c: 0 or aninteger of 1 or more; dN: deoxyribonucleotide and/or nucleotide analog;N: unmodified ribonucleotide and/or modified ribonucleotide, whereinsome of dNs in dNa may be replaced by Ns, and the nucleotide at the3′-terminus may be modified such that extension from the 3′-terminus bythe action of the DNA polymerase does not take place).

Such chimeric oligonucleotide primers are exemplified by a primer inwhich c is 0 and a primer in which the nucleotide analog isdeoxyriboinosine nucleotide or deoxyribouracil nucleotide, and themodified ribonucleotide is (α-S) ribonucleotide.

The composition of the thirty-first to thirty-fourth invention cancontain a buffering component suitable for a nucleic acid amplificationreaction. For example, it can contain a buffering component selectedfrom the group consisting of Bicine and HEPES.

The thirty-first to thirty-fourth invention is exemplified by acomposition containing a DNA polymerase selected from the groupconsisting of Klenow fragment of DNA polymerase I from Escherichia coli,Bst DNA polymerase lacking 5′3′ exonuclease from Bacillusstearothermophilus and Bca DNA polymerase lacking 5′3′ exonuclease fromBacillus caldotenax as the DNA polymerase having a strand displacementactivity. An endoribonuclease such as an RNase H can be used as theendonuclease. The RNase H is exemplified by an RNase H from Escherichiacoli, a bacterium of genus Thermotoga, a bacterium of genus Thermus, abacterium of genus Pyrococcus, a bacterium of genus Archaeoglobus, abacterium of genus Bacillus or the like.

In one aspect, the composition of the thirty-first to thirty-fourthinvention contains Bca DNA polymerase lacking 5′3′ exonuclease fromBacillus caldotenax as the DNA polymerase having a strand displacementactivity and an RNase H from Escherichia coli, a bacterium of genusPyrococcus or a bacterium of genus Archaeoglobus as the endonuclease.The RNase H is exemplified by type I RNase H from Escherichia coli, ortype II RNase H from a bacterium of genus Pyrococcus or a bacterium ofgenus Archaeoglobus.

The composition of the thirty-first to thirty-fourth invention maycontain a DNA polymerase having an endonuclease activity. Bca DNApolymerase lacking 5′3′ exonuclease from Bacillus caldotenax can be usedas such a DNA polymerase, which can be used in the presence of asubstance that allows the endonuclease activity of the DNA polymerase toexpress. The substance that allows the endonuclease activity of the BcaDNA polymerase to express is exemplified by a manganese ion.

The composition of the thirty-first to thirty-fourth invention cancontain a substance that inhibits the reverse transcription activity ofthe DNA polymerase. The substance that inhibits the reversetranscription activity of the DNA polymerase is exemplified byphosphonoformic acid. Furthermore, the composition may contain adeoxynucleotide triphosphate analog such as deoxyuridine triphosphate ora derivative thereof.

The thirty-fifth invention of the present invention relates to acomposition for amplifying a nucleic acid used for the method foramplifying a nucleic acid of the twenty-fourth to twenty-sixthinvention, which contains:

-   -   (a) an RNase H; and    -   (b) a DNA polymerase having a strand displacement activity.

The thirty-sixth invention of the present invention relates to acomposition for amplifying a nucleic acid used for the method foramplifying a nucleic acid of the twenty-seventh to thirtieth invention,which contains:

-   -   (a) an endonuclease; and    -   (b) a DNA polymerase having a strand displacement activity.

An endoribonuclease such as an RNase H can be used as the endonucleasefor the composition of the thirty-sixth invention.

An RNase H selected from the group consisting of an RNase H fromEscherichia coli, an RNase H from a bacterium of genus Thermotoga, anRNase H from a bacterium of genus Thermus, an RNase H from a bacteriumof genus Pyrococcus, an RNase H from a bacterium of genus Archaeoglobusand an RNase H from a bacterium of genus Bacillus can be used as theRNase H for the composition of the thirty-fifth or thirty-sixthinvention which contains an RNase H.

The composition of the thirty-fifth or thirty-sixth invention canfurther contain a buffering component suitable for a nucleic acidamplification reaction. For example, the composition may contain abuffering component selected from the group consisting of Bicine andHEPES.

The thirty-fifth or thirty-sixth invention is exemplified by acomposition that contains a DNA polymerase selected from the groupconsisting of Klenow fragment of DNA polymerase I from Escherichia coli,Bst DNA polymerase lacking 5′3′ exonuclease from Bacillusstearothermophilus and Bca DNA polymerase lacking 5′3′ exonuclease fromBacillus caldotenax as the DNA polymerase having a strand displacementactivity. An endoribonuclease such as an RNase H can be used as theendonuclease. The RNase H is exemplified by an RNase H from Escherichiacoli, a bacterium of genus Thermotoga, a bacterium of genus Thermus, abacterium of genus Pyrococcus, a bacterium of genus Archaeoglobus, abacterium of genus Bacillus or the like.

In one aspect, the composition of the thirty-fifth or thirty-sixthinvention contains Bca DNA polymerase lacking 5′3′ exonuclease fromBacillus caldotenax as the DNA polymerase having a strand displacementactivity and an RNase H from Escherichia coli, a bacterium of genusPyrococcus or a bacterium of genus Archaeoglobus as the endonuclease.The RNase H is exemplified by type I RNase H from Escherichia coli, ortype II RNase H from a bacterium of genus Pyrococcus or a bacterium ofgenus Archaeoglobus.

The composition of the thirty-fifth or thirty-sixth invention maycontain a DNA polymerase having an endonuclease activity. Bca DNApolymerase lacking 5′3′ exonuclease from Bacillus caldotenax can be usedas such a DNA polymerase, which can be used in the presence of asubstance that allows the endonuclease activity of the DNA polymerase toexpress. The substance that allows the endonuclease activity of the BcaDNA polymerase to express is exemplified by a manganese ion.

The composition of the thirty-fifth or thirty-sixth invention cancontain a substance that inhibits the reverse transcription activity ofthe DNA polymerase. The substance that inhibits the reversetranscription activity of the DNA polymerase is exemplified byphosphonoformic acid. Furthermore, the composition may contain adeoxynucleotide triphosphate analog such as deoxyuridine triphosphate ora derivative thereof.

The thirty-seventh invention of the present invention relates to a kitfor amplifying a nucleic acid used for the method for amplifying anucleic acid of the twenth-fourth to twenty-sixth of the presentinvention, which contains:

-   -   (a) an RNase H; and    -   (b) a DNA polymerase having a strand displacement activity.

The thirty-eighth invention of the present invention relates to a kitfor amplifying a nucleic acid used for the method for amplifying anucleic acid of the twenty-seventh to thirtieth invention of the presentinvention, which contains:

-   -   (a) an endonuclease; and    -   (b) a DNA polymerase having a strand displacement activity.

An endoribonuclease such as an RNase H can be used as the endonucleasefor the kit of the thirty-eighth invention.

The kit of the thirty-seventh or thirty-eighth invention which containsan RNase H is exemplified by a kit containing an RNase H selected fromthe group consisting of an RNase H from Escherichia coli, an RNase Hfrom a bacterium of genus Thermotoga, an RNase H from a bacterium ofgenus Thermus, an RNase H from a bacterium of genus Pyrococcus, an RNaseH from a bacterium of genus Archaeoglobus and an RNase H from abacterium of genus Bacillus.

The kit of the thirty-seventh or thirty-eighth invention may furthercontain a buffering component suitable for a nucleic acid amplificationreaction. For example, the composition can contain a buffering componentselected from the group consisting of Bicine and HEPES. The kit maycontain an annealing solution containing a substance that enhances theannealing of the nucleic acid as the template to the primer that issubstantially complementary to the nucleotide sequence of the nucleicacid. For example, the annealing solution may contain spermidine and/orpropylenediamine.

The DNA polymerase having a strand displacement activity contained inthe kit of the thirty-seventh or thirty-eighth invention of the presentinvention is exemplified by a DNA polymerase selected from the groupconsisting of Klenow fragment of DNA polymerase I from Escherichia coli,Bst DNA polymerase lacking 5′3′ exonuclease from Bacillusstearothermophilus and Bca DNA polymerase lacking 5′3′ exonuclease fromBacillus caldotenax.

In one aspect, the kit of the thirty-seventh or thirty-eighth inventioncontains Bca DNA polymerase lacking 5′3′ exonuclease from Bacilluscaldotenax and an RNase H from Escherichia coli, a bacterium of genusPyrococcus or a bacterium of genus Archaeoglobus. The RNase H isexemplified by type I RNase H from Escherichia coli, or type II RNase Hfrom a bacterium of genus Pyrococcus or a bacterium of genusArchaeoglobus.

The kit of the thirty-seventh or thirty-eighth invention of the presentinvention may contain a DNA polymerase having an endonuclease activity.Bca DNA polymerase lacking 5′3′ exonuclease from Bacillus caldotenax canbe used as such a DNA polymerase. The kit can contain a substance thatallows the endonuclease activity of the DNA polymerase to express. Thesubstance that allows the endonuclease activity of the Bca DNApolymerase to express is exemplified by a manganese ion.

The kit of the thirty-seventh or thirty-eighth invention can contain asubstance that inhibits the reverse transcription activity of the DNApolymerase. The substance that inhibits the reverse transcriptionactivity of the DNA polymerase is exemplified by phosphonoformic acid.Furthermore, the kit may contain a deoxynucleotide triphosphate analogsuch as deoxyuridine triphosphate or a derivative thereof.

The thirty-ninth invention of the present invention relates to a kit foramplifying a nucleic acid used for the method for amplifying a nucleicacid of the twenty-fourth to twenty-sixth invention of the presentinvention, which is in a packaged form and contains instructions thatdirect the use of a DNA polymerase having a strand displacement activityand an RNase H.

The fortieth invention of the present invention relates to a kit foramplifying a nucleic acid used for the method for amplifying a nucleicacid of the twenty-seventh to thirtieth invention of the presentinvention, which is in a packaged form and contains instructions thatdirect the use of a DNA polymerase having a strand displacement activityand an endonuclease.

The forty-first invention of the present invention relates to a productof a reagent for amplifying a nucleic acid consisting of a packingmaterial and a reagent for amplifying a nucleic acid enclosed in thepacking material, wherein the reagent for amplifying a nucleic acidcontains a DNA polymerase having a strand displacement activity and/oran RNase H, and description that the reagent for amplifying a nucleicacid can be used for nucleic acid amplification under isothermalconditions is indicated in a label stuck to the packaging material orinstructions attached to the packaging material.

The forty-second invention of the present invention relates to a productof a reagent for amplifying a nucleic acid consisting of a packingmaterial and a reagent for amplifying a nucleic acid enclosed in thepacking material, wherein the reagent for amplifying a nucleic acidcontains a DNA polymerase having a strand displacement activity and/oran endonuclease, and description that the reagent for amplifying anucleic acid can be used for nucleic acid amplification under isothermalconditions is indicated in a label stuck to the packaging material orinstructions attached to the packaging material.

The forty-third invention of the present invention relates to a methodfor detecting a target nucleic acid in a sample, characterized in thatthe method comprises:

-   -   (a) amplifying a nucleic acid by the method for amplifying a        nucleic acid of the twenty-fourth to thirtieth invention of the        present invention; and    -   (b) detecting a target nucleic acid amplified in step (a).

The detection method of the forty-third invention of the presentinvention can comprise detecting the amplified nucleic acid using aprobe for detection. The probe may be a probe that has been labeled witha labeling substance. For example, an RNA probe labeled with two or morefluorescent substances positioned at a distance that results in aquenching state can be used.

The forty-fourth invention of the present invention relates to achimeric oligonucleotide primer used for the forty-third invention. Thechimeric oligonucleotide primer is exemplified by a chimericoligonucleotide primer represented by general formula below:5′-dNa-Nb-dNc-3′  General formula(a: an integer of 11 or more; b: an integer of 1 or more; c: 0 or aninteger of 1 or more; dN: deoxyribonucleotide and/or nucleotide analog;N: unmodified ribonucleotide and/or modified ribonucleotide, whereinsome of dNs in dNa may be replaced by Ns, and the nucleotide at the3′-terminus may be modified such that extension from the 3′-terminus bythe action of the DNA polymerase does not take place).

Such chimeric oligonucleotide primers are exemplified by a primer inwhich c is 0 and a primer in which the nucleotide analog isdeoxyriboinosine nucleotide or deoxyribouracil nucleotide, and themodified ribonucleotide is (α-S) ribonucleotide.

The primer of the forty-fourth invention of the present invention isexemplified by a primer for detecting a pathogenic microorganism or adisease-related gene. A chimeric oligonucleotide primer for detecting apathogenic microorganism such as enterohemorrhagic Escherichia coli,Clostridium botulinum, Staphylococcus aureus, Mycobacteriumtuberculosis, Chlamydia trachomatis, papilloma virus, hepatitis C virusor a viroid is encompassed by the present invention.

The forty-fifth invention of the present invention relates to a chimericoligonucleotide primer for detecting enterohemorrhagic Escherichia colihaving a nucleotide sequence selected from the group consisting of SEQID NOS: 31 to 34, 47, 48, 51-53, 64-72, 84, 85, 113, 114, 130 and 131.

The forty-sixth invention of the present invention relates to a chimericoligonucleotide primer for detecting a viroid having a nucleotidesequence selected from the group consisting of SEQ ID NOS: 59, 60, 119,120, 122 and 123.

The forty-seventh invention of the present invention relates to achimeric oligonucleotide primer for detecting Clostridium botulinumhaving a nucleotide sequence represented by SEQ ID NO: 116 or 117.

The forty-eighth invention of the present invention relates to chimericoligonucleotide primer for detecting papilloma virus having a nucleotidesequence represented by SEQ ID NO: 96 or 97.

The forty-ninth invention of the present invention relates to a chimericoligonucleotide primer for detecting hepatitis C virus having anucleotide sequence selected from the group consisting of SEQ ID NOS:101, 102, 138, 139, 200, 201, 205 and 206.

The fiftieth invention of the present invention relates to a chimericoligonucleotide primer for detecting Staphylococcus aureus having anucleotide sequence represented by SEQ ID NO: 136 or 137.

The fifty-first invention of the present invention relates to a chimericoligonucleotide primer for detecting Mycobacterium tuberculosis having anucleotide sequence selected from the group consisting of SEQ ID NOS:155, 156, 159 to 162, 194 and 195.

The fifty-second invention of the present invention relates to achimeric oligonucleotide primer for detecting Chlamydia trachomatishaving a nucleotide sequence selected from the group consisting of SEQID NOS: 157, 158, 203 and 204.

The fifty-third invention of the present invention relates to a kit foramplifying a nucleic acid used for the method for amplifying a nucleicacid of the twenty-fourth to twenty-eighth invention of the presentinvention, which contains the chimeric oligonucleotide primer of theforty-fourth to fifty-second invention.

The fifty-fourth invention of the present invention relates to a kit fordetecting a target nucleic acid used for the method for detecting atarget nucleic acid of the forty-third invention of the presentinvention, which contains the chimeric oligonucleotide primer of theforty-fourth to fifty-second invention.

The fifty-fifth invention of the present invention relates to a probeused in the method of the forty-third invention.

The fifty-sixth invention of the present invention relates to a probewhich hybridizes to the nucleic acid amplified by the method of thetwenty-third to thirtieth invention.

The fifty-seventh invention of the present invention relates to a probewhich hybridizes to a region amplified using the chimericoligonucleotide primer of the forty-fourth to fifty-second invention.

The probe of the fifty-fifth to fifty-seventh invention may be a probewhich has been labeled with a labeling substance, for example, an RNAprobe labeled with two or more fluorescent substances positioned at adistance that results in a quenching state

The fifty-eighth invention of the present invention relates to a kitused for the detection of the target nucleic acid in the method of theforty-third invention, which contains the probe of the fifty-fifth tofifty-seventh invention.

The fifty-ninth invention of the present invention relates to a methodfor amplifying a nucleic acid, which comprises using a DNA polymerasehaving a strand displacement activity to effect a template switchingreaction.

The DNA polymerase having a strand displacement activity used in thefifty-ninth invention is exemplified by Klenow fragment of DNApolymerase I from Escherichia coli, Bst DNA polymerase lacking 5′3′exonuclease from Bacillus stearothermophilus and Bca DNA polymeraselacking 5′3′ exonuclease from Bacillus caldotenax.

The sixtieth invention of the present invention relates to a method forproducing a material having an immobilized nucleic acid in which thenucleic acid is arrayed in a predefined region, characterized in thatthe method comprises:

-   -   (a) amplifying a nucleic acid to be immobilized by the method        for amplifying a nucleic acid of the twenty-fourth to thirtieth        invention of the present invention; and    -   (b) arraying and immobilizing the nucleic acid amplified in        step (a) in a predefined region.

The sixty-first invention of the present invention relates to a materialhaving an immobilized nucleic acid in which the nucleic acid is arrayedin a predefined region produced by the method of the sixtieth invention.

The sixty-second invention of the present invention relates to a methodfor producing a nucleic acid in large quantities, characterized in thatthe method comprises:

-   -   (a) amplifying a nucleic acid by the method for amplifying a        nucleic acid of the twenty-fourth to thirtieth invention of the        present invention; and    -   (b) collecting the nucleic acid amplified in step (a).

The sixty-third invention of the present invention relates to a methodfor amplifying a nucleic acid, characterized in that the methodcomprises:

-   -   (a) duplicating a DNA or an RNA containing a sequence to be        amplified to prepare a nucleic acid as a template; and    -   (b) amplifying the nucleic acid as the template obtained in        step (a) by the method for amplifying a nucleic acid of the        twenty-fourth to thirtieth invention.

The sixty-fourth invention of the present invention relates to a methodfor determining a nucleotide sequence of a nucleic acid, characterizedin that the method comprises amplifying a nucleic acid according to themethod of the twenty-fourth to thirtieth, sixty-second or sixty-thirdinvention.

The sixty-fifth invention of the present invention relates to a methodfor preparing a single-stranded nucleic acid, the method comprising astep of generating a single-stranded nucleic acid using the methodaccording to the twenty-fourth to thirtieth invention. In thesixty-fifth invention, at least two primers at different concentrationsmay be used.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 2 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 3 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 4 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 5 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 6 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 7 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 8 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 9 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 10 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 11 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 12 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 13 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 14 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 15 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 16 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 17 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 18 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 19 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 20 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 21 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 22 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 23 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 24 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 25 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 26 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 27 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 28 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention andthe results of dot blot hybridization.

FIG. 29 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 30 is a graph which compares the amounts of amplification productsamplified according to the method of the present invention and the PCR.

FIG. 31 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 32 is a photograph of polyacrylamide gel electrophoresis ofamplified DNA fragments amplified according to the method of the presentinvention.

FIG. 33 is a figure illustrating one aspect of the method for amplifyinga nucleic acid of the present invention.

FIG. 34 is a figure illustrating one aspect of the method for amplifyinga nucleic acid of the present invention.

FIG. 35 is a figure illustrating one aspect of the method for amplifyinga nucleic acid of the present invention.

FIG. 36 is a figure illustrating one aspect of the method for amplifyinga nucleic acid of the present invention.

FIG. 37 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 38 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 39 is a photograph of agarose gel electrophoresis of amplified DNAfragments amplified according to the method of the present invention.

FIG. 40 is a flow chart that illustrates an example of the method of thepresent invention in which a single-stranded DNA is used. In the figure,the released DNA strand marked with the closed circle serves as atemplate DNA in (6).

FIG. 41 shows the results of agarose gel electrophoresis of DNAfragments amplified by the method of the present invention using varyingreaction time.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a deoxyribonucleotide (also referred to as a dN) refersto a nucleotide of which the sugar portion is composed ofD-2-deoxyribose. The deoxyribonucleotides include, for example, oneshaving adenine, cytosine, guanine or thymine as the base portion.Furthermore, the doxyribonucleotides also include a deoxyribonucleotidehaving a modified base such as 7-deazaguanosine and adeoxyribonucleotide analog such as deoxyinosine nucleotide.

As used herein, a ribonucleotide (also referred to as an N) refers to anucleotide of which the sugar portion is composed of D-ribose. Theribonucleotides include ones having adenine, cytosine, guanine or uracilas the base portion. The ribonucleotides also include modifiedribonucleotides such as a modified ribonucleotide in which the oxygenatom of the phosphate group at the α-position is replaced by a sulfuratom (also referred to as an (α-S) ribonucleotide or an (α-S)N) or otherderivatives.

As used herein, a chimeric oligonucleotide primer refers to a primerthat contains a deoxyribonucleotide and a ribonucleotide. The primer maycontain a nucleotide analog and/or a modified ribonucleotide.

The chimeric oligonucleotide primers used in the present inventioninclude any chimeric oligonucleotide primer that has a ribonucleotidebeing positioned at the 3′-terminus or on the 3′-terminal side of theprimer, can be used to extend a nucleic acid strand in the method of thepresent invention, can be cleaved with an endonuclease, and can be usedto effect a strand displacement reaction.

As used herein, 3′-terminal side refers to a portion from the center tothe 3′-terminus of a nucleic acid such as a primer. Likewise,5′-terminal side refers to a portion from the center to the 5′-terminusof a nucleic acid.

As used herein, an endonuclease may be any one that acts on adouble-stranded DNA generated by extending a DNA from the chimericoligonucleotide primer which have been annealed to a nucleic acid as atemplate, and specifically cleaves it at a portion of the primer thatcontains a ribonucleotide.

As used herein, a DNA polymerase refers to an enzyme that synthesizes aDNA strand de novo using a DNA strand as a template. The DNA polymerasesinclude naturally occurring DNA polymerases and variant enzymes havingthe above-mentioned activity. For example, such enzymes include a DNApolymerase having a strand displacement activity, a DNA polymeraselacking a 5′3′ exonuclease activity and a DNA polymerase having areverse transcriptase activity or an endonuclease activity.

As used herein, “a strand displacement activity” refers to an activitythat can effect a strand displacement, that is, that can proceed DNAduplication on the basis of the sequence of the nucleic acid as thetemplate while displacing the DNA strand to release the complementarystrand that has been annealed to the template strand. In addition, a DNAstrand released from a nucleic acid as a template as a result of astrand displacement is referred to as “a displaced strand” herein.

Hereinafter, the present invention will be described in detail.

(1) Chimeric Oligonucleotide Primer Used in the Present Invention.

The primer used in the method of the present invention is a chimericoligonucleotide primer that contains a ribonucleotide as well as atleast one selected from the group consisitng of a deoxyribonucleotideand a nucleotide analog. Such primers also include anoligoribonucleotide primer that contains an unmodified ribonucleotideand/or a modified ribonucleotide.

A chimeric oligonucleotide primer used in the method of the presentinvention is a chimeric oligonucleotide primer that has a nucleotidesequence substantially complementary to a part of the nucleotidesequence of a nucleic acid as a template. It can contribute to extensionof a DNA strand under conditions used. Furthermore, a ribonucleotide ispositioned at the 3′-terminus or on the 3′-terminal side of the chimericoligonucleotide primer. The primer is usually designed such that it iscomplementary to a portion upstream of the region to be amplified, thatis, a portion 3′ to the nucleotide sequence corresponding to a region tobe amplified in a nucleic acid as a template. As used herein, “asubstantially complementary nucleotide sequence” means a nucleotidesequence that can anneal to a DNA as a template under reactionconditions used.

The chimeric oligonucleotide primer used in the method of the presentinvention may contain one or more modified ribonucleotide. As usedherein, a ribonucleotide may be an unmodified ribonucleotide and/or amodified ribonucleotide that can be positioned at the 3′-terminus or onthe 3′-terminal side of a chimeric oligonucleotide primer and that isrecognized by or cleaved with an endonuclease. The ribonucleotidesinclude both of the unmodified ribonucleotide and the modifiedribonucleotide as described above. An unmodified ribonucleotide, amodified ribonucleotide or a combination thereof can be used for thechimeric oligonucleotide primer of the present invention as long as itdoes not abolish the function of the primer. Examples of the modifiedribonucleotides include, but are not limited to, an (α-S) ribonucleotidein which the oxygen atom bound to the phosphate group is replaced by asulfur atom, and a ribonucleotide in which the hydroxy group at the2-position of the ribose is replaced by a methoxy group. Such a chimericoligonucleotide primer containing a modified ribonucleotide can beproduced by using, for example, an (α-S) ribonucleotide triphosphate,which is prepared by a method using a sulfuration reaction reagent (GlenResearch) as described in U.S. Pat. No. 5,003,097, or a 2-OMe-RNA-CEphosphoramidite reagent (Glen Research).

A chimeric oligonucleotide primer that can be used in the amplificationmethod of the present invention may be designed to contain a modifiedribonucleotide that confers resistance to the cleavage with anendonuclease. Such a primer is useful in that one can control thecleavage site with an endonuclease during amplification reaction steps.

One or two chimeric oligonucleotide primer may be used in the method ofthe present invention depending on the desired form of a DNA fragmentafter amplification (single-stranded or double-stranded). Specifically,one chimeric oligonucleotide primer is used when a single-stranded DNAis desired, whereas two primers are used when a double-stranded DNA isdesired.

The length of the chimeric oligonucleotide primer used in the method ofthe present invention is not specifically limited, but is preferablyabout 12 nucleotides to about 100 nucleotides, more preferably about 15nucleotides to about 40 nucleotides. It is preferable that thenucleotide sequence of the chimeric oligonucleotide is substantiallycomplementary to a nucleic acid as a template such that it anneals tothe nucleic acid as the template under reaction conditions used. Theprimer contains a sequence recognized by an endonuclease, which isutilized in a step as described below, at the 3′-terminus or on the3′-terminal side.

For example, an oligonucleotide having a structure represented by thefollowing general formula can be used in the DNA synthesis method of thepresent invention as a primer, although it is not intended to limit thepresent invention:5′-dNa-Nb-dNc-3′  General formula(a: an integer of 11 or more; b: an integer of 1 or more; c: 0 or aninteger of 1 or more; dN: deoxyribonucleotide and/or nucleotide analog;N: unmodified ribonucleotide and/or modified ribonucleotide, whereinsome of dNs in dNa may be replaced by Ns, and the nucleotide at the3′-terminus may be modified such that extension from the 3′-terminus bythe action of the DNA polymerase does not take place).

For example, a chimeric oligonucleotide primer represented by thegeneral formula in which a=an integer of 11 or more; and b=1 and c=0,b=2 and c=0, b=3-5 and c=0, or b=2 and c=0-5 can be preferably used inthe present invention. The length of the ribonucleotides at the3′-terminus or on the 3′-termunal side of the chimeric oligonucleotideprimer used in the method of the present invention is preferably 1-merto 15-mer, more preferably 1-mer to 10-mer, most preferably 1-mer to5-mer. The number of c in the general formula is not specificallylimited, but any number that can be used in the method of the presentinvention may be selected. Usually, 5 or less is preferably. Betterresults are obtained in a reaction by selecting 3 rather than 4, 2rather than 3, and 1 rather than 2 for c. In particular, the mostefficient reaction can be accomplished in case of c=0.

The chimeric oligonucleotide primer used in the present invention has astructure in which an endonuclease recognizes or cleaves a DNA strandextended from the primer using a DNA polymerase (a primer-extendedstrand) at a site that contains a ribonucleotide, which ribonucleotidebeing positioned at the 3′-terminus or on the 3′-terminal side of thechimeric oligonucleotide primer. Although it is not intended to limitthe present invention, for example, when an RNase H acts on adouble-stranded DNA generated by extending a DNA from a chimericoligonucleotide primer represented by the general formula that has beenannealed to a nucleic acid as a template, the chimeric oligonucleotideprimer is cleaved at the ribonucleotide portion. A double-stranded DNAin which a nick is introduced between the oligonucleotide primer and theDNA strand synthesized by the extension is then generated. Then, astrand displacement reaction with a DNA polymerase proceeds from thenicked site. Thus, any chimeric oligonucleotide primer that can be usedto extend a nucleic acid strand from the 3′-terminus of the primer, thatcan be cleaved with an endonuclease, and with which a DNA polymerase caneffect a strand displacement reaction can be used in the method of thepresent invention. Furthermore, the chimeric oligonucleotide primers ofthe present invention include one whose 3′-terminus is modified suchthat extension by the action of the DNA polymerase can not take place,and DNA extension takes place from a 3′-terminus generated upon cleavageby the endonuclease. In addition, a promoter sequence for an RNApolymerase may be included on the 5′-terminal side of the chimericoligonucleotide primer. Such RNA polymerases are exemplified by T7 RNApolymerase and SP6 RNA polymerase.

Furthermore, the chimeric oligonucleotide primer used in the method ofthe present invention may contain a nucleotide analog or othersubstances. That is, one or more nucleotide analog(s) can be containedin the chimeric oligonucleotide primer of the present invention as longas the function of the primer is not abolished. Plural types of thenucleotide analogs can be used in combination. Examples of thenucleotide analogs include, but are not limited to, nucleotide analogshaving ribose derivatives such as deoxyinosine nucleotide, deoxyuracilnucleotide and a deoxyribonucleotide analog having a modified base suchas 7-deazaguanine. Furthermore, the chimeric oligonucleotides used inthe present invention may contain deoxynucleotides, ribonucleotides ornucleotide analogs having various modifications such as addition oflabeled compounds as long as they retain the functions as describedabove.

Incorporation of a nucleotide analog into a primer is effective forsuppressing the formation of high-order structure of the primer itselfand stabilization of annealing formation with the template. Aribonucleotide may be incorporated into a primer for the same purpose.Although it is not intended to limit the present invention, a modifiedribonucleotide such as (α-S) ribonucleotide can be preferably used inorder to prevent the digestion of the primer by a non-specificendonuclease (RNase).

The chimeric oligonucleotide primer can be synthesized to have desirednucleotide sequence using, for example, the 394 type DNA synthesizerfrom Applied Biosystems Inc. (ABI) according to a phosphoramiditemethod. Alternatively, any methods including a phosphate triestermethod, an H-phosphonate method and a thiophosphonate method may be usedto synthesize the chimeric oligonucleotide primer.

(2) Endonuclease Used in the Present Invention.

Any endonuclease that can act on a double-stranded DNA generated by DNAextension from the chimeric oligonucleotide primer as described above in(1) that has been annealed to a nucleic acid as a template and cleavesthe extended strand to effect a strand displacement reaction may be usedin the present invention. That is, the endonuclease is an enzyme thatcan generate a nick in the chimeric oligonucleotide primer portion ofthe double-stranded DNA. Examples of endonucleases that can be used inthe present invention include, but are not limited to, ribonucleases.Among these, endoribonuclease H (RNase H) that acts on an RNA portion ofa double-stranded nucleic acid composed of a DNA and an RNA can bepreferably used. Any ribonuclease that has the above-mentionedactivities can be preferably used in the present invention, includingmesophilic and heat-resistant ones. For example, an RNase H from E. colican be used for a reaction at about 50 C to about 70 C in the method ofthe present invention as described below in Examples. A heat-resistantribonuclease can be preferably used in the method of the presentinvention. Examples of the heat-resistant ribonucleases which can bepreferably used include, but are not limited to, a commerciallyavailable ribonuclease, Hybridase™ Thermostable RNase H (EpicenterTechnologies) as well as an RNase from a thermophilic bacterium of genusBacillus, a bacterium of genus Thermus, a bacterium of genus Pyrococcus,a bacterium of genus Thermotoga, a bacterium of genus Archaeoglobus orthe like. Furthermore, both of naturally occurring ribonucleases andvariants can be preferably used. The enzymatic unit of RNase H indicatedherein is a value expressed according to a method of measuring anenzymatic unit as described in Referential Examples.

The RNase H is not limited to a specific one as long as it can be usedin the method of the present invention. For example, the RNase H may bederived from any organisms including various viruses, phages,prokaryotes and eukaryotes. It may be either a bacterial RNase H or aviral RNase H. The bacterial RNase H is exemplified by Escherichia coliRNase H I and the viral RNase H is exemplified by HIV-1. Type I, type IIor type III RNase H can be used in the method of the present invention.For example, type I RNase H from Escherichia coli, or type II RNase Hfrom a bacterium of genus Pyrococcus or a bacterium of genusArchaeoglobus is preferable, without limitation.

The efficiency of the cleavage reaction with an endonuclease such asRNase H used in the method of the present invention may vary dependingon the nucleotide sequence around the 3′-terminus of the primer andinfluence the amplification efficiency of the desired DNA. Therefore, itis natural to design the optimal primer for the RNase H used.

As used herein, the term “introducing a nick” or “nicking” meansinternally cleaving one of the two strands of a double-stranded nucleicacid. For example, an RNase H acts on a hybrid double-stranded nucleicacid composed of a DNA and a ribonucleotide-containing DNA toselectively cleave the ribonucleotide-containing strand among the twostrands at the ribonucleotide portion, thereby introducing a nick intothe hybrid double-stranded nucleic acid.

(3) DNA Polymerase Used in the Present Invention.

A DNA polymerase having a strand displacement activity on a DNA can beused in the present invention. Particularly, a DNA polymerasesubstantially lacking a 5′3′ exonuclease activity can be preferablyused.

As used herein, “a strand displacement activity” refers to an activitythat can effect a strand displacement, that is, that can proceed DNAduplication on the basis of a sequence of a nucleic acid as a templatewhile displacing a DNA strand to release a complementary strand that hasbeen annealed to the template strand. Additionally, a DNA strandreleased from a nucleic acid as a template as a result of a stranddisplacement is referred to as “a displaced strand” herein.

Any DNA polymerases having the strand displacement activity can be usedin the present invention. Examples thereof include variants of DNApolymerases lacking their 5′3′ exonuclease activities derived fromthermophilic bacteria of genus Bacillus such as Bacillus caldotenax(hereinafter referred to as B. ca) and Bacillus stearothermophilus(hereinafter referred to as B. st), as well as large fragment (Klenowfragment) of DNA polymerase I from Escherichia coli (E. coli). Both ofmesophilic and heat-resistant DNA polymerases can be preferably used inthe present invention.

B. ca is a thermophilic bacterium having an optimal growth temperatureof about 70° C. Bca DNA polymerase from this bacterium is known to havea DNA-dependent DNA polymerase activity, an RNA-dependent DNA polymeraseactivity (a reverse transcription activity), a 5′3′ exonuclease activityand a 3′ 5′ exonuclease activity. The enzyme may be either an enzymepurified from its original source or a recombinant protein produced byusing genetic engineering techniques. The enzyme may be subjected tomodification such as substitution, deletion, addition or insertion byusing genetic engineering techniques or other means. Examples of suchenzymes include BcaBEST DNA polymerase (Takara Shuzo), which is Bca DNApolymerase lacking its 5′3′ exonuclease activity.

It is known that some DNA polymerases have an endonuclease activity suchas an RNase H activity under specific conditions. Such a DNA polymerasecan be used in the method of the present invention. In one aspect, theDNA polymerase may be used under conditions that allow the RNase Hactivity to express, e.g., in the presence of Mn²⁺. In this case, themethod of the present invention can be conducted without the addition ofan RNase H. The present inventors have demonstrated that the Bca DNApolymerase exhibits an RNase activity in a buffer containing Mn²⁺ forthe first time, and that the method for amplifying a nucleic acid of thepresent invention can be carried out in a reaction mixture containing noenzyme other than the Bca DNA polymerase. The above-mentioned aspect isnot limited to the use of the Bca DNA polymerase. DNA polymerases thatare known to have an RNase H activity such as Tth DNA polymerase fromThermus thermophilus can be used in the present invention.

(4) Composition of Reaction Buffer Used in the Present Invention.

A reaction buffer that contains a buffering component, a magnesium saltor another metal salt and dNTPs is used in the present invention.Naturally, the type and the concentration of the salt are optimizeddepending on the metal requirement or the like of the enzyme to be used.Examples of the buffering components that can be preferably usedinclude, but are not limited to, Bicine, Tricine, HEPES, tris and aphosphate (such as sodium phosphate and potassium phosphate). Amongthese, a buffer that contains Bicine, Tricine, HEPES or a phosphate as abuffering component is preferable for the present invention. Although itis not intended to limit the present invention, for example, when thereaction temperature is high, a Bicine buffer of which the change in pHdue to the change in temperature is little is preferably used. A HEPESbuffer may be preferable depending on the type of RNase H used. Thus,the optimal buffer may be selected depending on the reactiontemperature, the endonuclease or the DNA polymerase to be used and thelike. The final concentration of the buffering component ranges 5-100mM, preferably 20-50 mM. The pH ranges 6.0-9.5, preferably 7.0-9.2. Forexample, a buffer containing 22-46 mM Tricine at pH 7.5-9.2 or a buffercontaining 25-50 mM potassium phosphate at pH 7.0-8.0 is preferablyused. Examples of magnesium salts that can be preferably used include,but are not limited to, magnesium chloride, magnesium acetate ormagnesium sulfate. The final concentration of the magnesium salt ranges1-20 mM, preferably 2-10 mM. The final concentrations of dNTPs (amixture of DATP, dCTP, dGTP and dTTP) in a mixture as substrates for aDNA extension reaction range 0.1-3.0 mM, preferably 0.2-1.2 mM. Theamount of the primers used in a reaction volume of 50 μl ranges 1-1000pmol, preferably 10-150 pmol. Additionally, the reaction mixture maycontain an additive, for example, in order to stabilize theamplification reaction. Bovine serum albumin (BSA) at a finalconcentration of 0.1% or less, dimethyl sulfoxide (DMSO) at a finalconcentration of 10% or less, putrescine dihydrochloride at a finalconcentration of 4 mM or less, or propylenediamine at a finalconcentration of 0.01% or less may be added. Additionally, NMP(1-methyl-2-pyrrolidinone), glycerol, polyethylene glycol, dimethylsulfoxide and/or formamide may be contained. It is expected that theaddition of such an organic solvent reduces the non-specific annealingof oligonucleotide primers.

The method of the present invention may be carried out by adding asubstance that inhibits the reverse transcription activity of the DNApolymerase such as phosphonoformic acid (PFA). If a substance thatinhibits the reverse transcription activity is added, the amplificationof non-specific products other than the target nucleic acid is reduced.

In another aspect, annealing of the nucleic acid as the template to thechimeric oligonucleotide primer used in the present invention before theamplification reaction is effective to reduce non-specific annealing ofan oligonucleotide primer in the detection, amplification or productionmethod of the present invention. It is preferable to conduct theannealing using an annealing solution that contains a substance thatenhances the annealing such as a polyamine (e.g., spermine orspermidine) or propylenediamine. Preferably, the annealing solutioncontaining the polyamine also contains a salt. For example, withoutlimitation, the annealing solution may contain sodium chloride,potassium chloride, potassium acetate, sodium acetate or the like and apolyamine.

The annealing is usually conducted by incubating the annealing solutioncontaining the primer and the nucleic acid as the template at atemperature at which a double-stranded nucleic acid is denatured (e.g.,90 C or above) and then cooling the solution to a reaction temperatureused for the method of the present invention or below.

After annealing, the nucleic acid amplification reaction of the presentinvention is initiated by further adding other necessary components suchas a DNA polymerase, an RNase H and dNTPs to the mixture.

The amount of an RNase H from Escherichia coli as an example ofendonucleases in a reaction volume of 50 μl ranges preferably 3-200 U,more preferably 15-60 U. Similarly, the amount of an RNase H from abacterium of genus Pyrococcus or a bacterium of genus Archaeoglobus asan example of endonuclease in a reaction volume of 50 μl rangespreferably 3-200 U, more preferably 4-40 U. The amount of BcaBEST DNApolymerase (Takara Shuzo) as an example of DNA polymerases in a reactionvolume of 50 μl ranges preferably 0.5-100 U, more preferably 1-22 U.

When an endonuclease and a DNA polymerase is used in combination in themethod of the present invention, for example, without limitation, acombination of an RNase H from Escherichia coli, a bacterium of genusPyrococcus or a bacterium of genus Archaeoglobus and BcaBEST DNApolymerase is preferably used. It is considered that the preferableunits of the endonuclease and the DNA polymerase may vary depending onthe types the enzymes. In such a case, the composition of the bufferused and the amount of the enzymes added may be adjusted using theincrease in detection sensitivity or the amount of amplification productas an index. In either case, it is natural to optimize the compositionof the reaction buffer and the like depending on the type of the enzymeto be used.

(5) Method for Amplifying Nucleic Acid of the Present Invention.

The method of the present invention can be conducted by using at leastone oligonucleotide primer as described above in (1) in combination withthe endonuclease as described above in (2) and the DNA polymerase asdescribed above in (3). Alternatively, a DNA polymerase having an RNaseH activity can be used under conditions that allow the RNase H activityto express as described above.

dNTPs used for the PCR or the like (a mixture of DATP, dCTP, dGTP anddTTP) can be preferably used as nucleotide triphosphates that serve assubstrates in the extension reaction in the method. In addition, dUTPmay be used as a substrate. The dNTPs may contain a dNTP(deoxyribonucleotide triphosphate) analog such as 7-deaza-dGTP,triphosphate of dITP or the like as long as it serves as a substrate forthe DNA polymerase used. A derivative of a DNTP or a dNTP analog may beused. A derivative having a functional group such as a dUTP having anamino group may be contained. A chimeric oligonucleotide primer is usedin the method. The primer can be prepared, for example, using a DNAsynthesizer according to a conventional synthesis method. A combinationof the chimeric oligonucleotide primer and a normal oligonucleotideprimer can be used in the method of the present invention.

If the activity of the enzyme used may be decreased in the course of thereaction, the enzyme can be further added during the reaction in themethod of the present invention. Although it is not intended to limitthe present invention, for example, an RNase H from Escherichia coli maybe further added during a reaction in which the RNase H is used. Theadded enzyme may be the same as that contained in the reaction mixtureat the beginning of the reaction or it may be a different enzyme thatexhibits the same activity. Thus, the type or the property of the enzymeto be added is not limited to a specific one as long as the additionduring the reaction provides effects such as increase in the detectionsensitivity or increase in the amount of amplification product.

The nucleic acid (DNA or RNA) used as a template in the method of thepresent invention may be prepared or isolated from any sample that maycontain the nucleic acid. Alternatively, the sample may be used directlyin the method for amplifying a nucleic acid of the present invention.Examples of the samples that may contain the nucleic acid include, butare not limited to, samples from organisms such as a whole blood, aserum, a buffy coat, a urine, feces, a cerebrospinal fluid, a seminalfluid, a saliva, a tissue (e.g., a cancerous tissue or a lymph node) anda cell culture (e.g., a mammalian cell culture or a bacterial cellculture), samples that contain a nucleic acid such as a viroid, a virus,a bacterium, a fungi, a yeast, a plant and an animal, samples suspectedto be contaminated or infected with a microorganism such as a virus or abacterium (e.g., a food or a biological formulation), and samples thatmay contain an organism such as a soil and a waste water. The sample maybe a preparation containing a nucleic acid obtained by processing theabove-mentioned samples according to a known method. Examples of thepreparations that can be used in the present invention include a celldestruction product or a sample obtained by fractionating the product,the nucleic acid in the sample, or a sample in which specific nucleicacid molecules such as mRNAs are enriched. Furthermore, a nucleic acidsuch as a DNA or an RNA obtained amplifying a nucleic acid contained inthe sample by a known method can be preferably used.

The preparation containing a nucleic acid can be prepared from theabove-mentioned materials by using, for example, lysis with a detergent,sonication, shaking/stirring using glass beads or a French press,without limitation. In some cases, it is advantageous to further processthe preparation to purify the nucleic acid (e.g., in case where anendogenous nuclease exists). In such cases, the nucleic acid is purifiedby a know method such as phenol extraction, chromatography, ionexchange, gel electrophoresis or density-gradient centrifugation.

When it is desired to amplify a nucleic acid having a sequence derivedfrom an RNA, the method of the present invention may be conducted usingas a template a cDNA synthesized by a reverse transcription reactionthat uses the RNA as a template. Any RNA for which one can make a primerto be used in a reverse transcription reaction can be applied to themethod of the present invention, including total RNA in a sample, RNAmolecules such as, mRNA, tRNA and rRNA as well as specific RNA molecularspecies.

Any primer that anneals to an RNA as a template under reactionconditions used can be used in the reverse transcription reaction. Theprimer may be a primer having a nucleotide sequence that iscomplementary to a specific RNA as a template (a specific primer), anoligo-dT (deoxythymine) primer and a primer having a random sequence (arandom primer). In view of specific annealing, the length of the primerfor reverse transcription is preferably 6 nucleotides or more, morepreferably 9 nucleotides or more. In view of oligonucleotide synthesis,the length is preferably 100 nucleotides or less, more preferably 30nucleotides or less. A chimeric oligonucleotide primer can be used as aprimer for reverse transcription. The chimeric oligonucleotide primercan also be utilized as a primer for a strand displacement reaction inthe method for amplifying a nucleic acid of the present invention usinga cDNA obtained after reverse transcription as a template. Such a primermay be any one that has the properties as described above in (1) andthat can be used in a reverse transcription reaction from an RNA.

Any enzyme that has an activity of synthesizing a cDNA using an RNA as atemplate can be used in the reverse transcription reaction. Examplesthereof include reverse transcriptases originating from various sourcessuch as avian myeloblastosis virus-derived reverse transcriptase (AMVRTase), Molony murine leukemia virus-derived reverse transcriptase (MMLVRTase) and Rous-associated virus 2 reverse transcriptase (RAV-2 RTase).In addition, a DNA polymerase that also has a reverse transcriptionactivity can be used. An enzyme having a reverse transcription activityat a high temperature such as a DNA polymerase from a bacterium of genusThermus (e.g., Tth DNA polymerase) and a DNA polymerase from athermophilic bacterium of genus Bacillus is preferable for the presentinvention. For example, DNA polymerases from thermophilic bacteria ofgenus Bacillus such as a DNA polymerase from B. st (Bst DNA polymerase)and Bca DNA polymerase are preferable, although it is not intended tolimit the present invention. For example, Bca DNA polymerase does notrequire a manganese ion for the reverse transcription reaction.Furthermore, it can synthesize a cDNA while suppressing the formation ofa secondary structure of an RNA as a template under high-temperatureconditions. Both a naturally occurring one and a variant of the enzymehaving a reverse transcriptase activity can be used as long as they havethe activity.

In another aspect, after duplicating a DNA or an RNA containing anucleotide sequence to be amplified beforehand, the duplicated productmay be used as a nucleic acid as a template in the method of the presentinvention. Examples of the methods for duplication include, but are notlimited to, a method in which an appropriate host is transformed with avector into which the nucleic acid fragment containing the nucleotidesequence to be amplified is inserted, the resulting transformant iscultured, the vector into which the nucleic acid fragment containing thenucleotide sequence to be amplified is inserted is extracted therefromand used. Any vectors can be used as long as they are stably replicatedin the host. For example, pUC series, pBluescript series, pGEM series,cosmid type vectors and phage type vectors are preferably used. Anyhosts can be used as long as they can maintain the vectors used. Forexample, Escherichia coli, which is readily cultured, is exemplified.

In another aspect of the duplication method, after an RNA having anucleotide sequence to be amplified is transcribed using an RNApolymerase and a nucleic acid fragment containing the nucleotidesequence as a template, the RNA may be used as a template for the methodof the present invention directly or after converting it into a cDNA byreverse transcription reaction. The nucleic acid fragment containing thenucleotide sequence to be amplified is not limited to a specific one aslong as it has a promoter sequence for an RNA polymerase. It may beinserted into a vector having a promoter sequence for an RNA polymerase,ligated with an adapter or a cassette having a promoter sequence for anRNA polymerase at its end or enzymatically synthesized using a primerhaving a promoter sequence for an RNA polymerase and an appropriatetemplate. Thus, a nucleic acid fragment containing a nucleotide sequenceto be amplified can be duplicated or amplified in a form of an RNA usinga promoter sequence for an RNA polymerase being positioned as describedabove. Any vectors can be used as long as they have promoter sequencesfor RNA polymerases. For example, pUC series, pBluescript series, pGEMseries, cosmid type vectors and phage type vectors are preferably used.The vector can be preferably used in its circular form or after beinglinearized. Any RNA polymerases can be used for the duplication oramplification method. For example, SP6 RNA polymerase, T7 RNApolymerase, T3 RNA polymerase or the like can be preferably used.

Both of a double-stranded DNA such as a genomic DNA isolated asdescribed above or a PCR fragment and a single-stranded DNA such as acDNA prepared by a reverse transcription reaction from a total RNA or anmRNA can be preferably used as a template DNA in the present invention.The double-stranded DNA is preferably used either after denaturing itinto single-stranded DNAs or without the denaturation.

The denaturing step may be eliminated in the nucleic acid amplificationmethod of the present invention if a linear double-stranded DNA such asa PCR amplification product is used as a template. The elimination maybe accomplished by locating the annealing site for the primer used inthe method of the present invention about 50 bases inside from theterminus of the DNA. If a nucleic acid having a sequence from an RNA isto be amplified, amplification reaction can be initiated by adding anRNA-cDNA double-stranded nucleic acid obtained by reverse transcriptionreaction using an RNA as a template to the amplification reactionmixture of the present invention containing an RNase H to digest the RNAstrand and convert the nucleic acid into a single-stranded cDNA.Furthermore, a reverse transcription reaction using an RNA as a templateand a DNA amplification reaction using a cDNA generated by the reversetranscription reaction as a template can be conducted with one DNApolymerase in the DNA synthesis method of the present invention. Such aDNA polymerase has a reverse transcriptase activity and a stranddisplacement activity.

The suitable length of the template is one that provides a sufficientbinding of the primer sequence due to the presence of the whole targetsequence or at least a sufficient part of the target sequence in thefragment.

Without limitation, if a DNA as a template is a double-stranded DNA, theDNA can be denatured into single-stranded DNAs to allow a primer to bindto the DNA strand as the template in the method of the presentinvention. Incubating the double-stranded DNA at a temperature at whichit is denatured (e.g., about 95° C.) is preferable for the denaturation.Other processes include one in which an elevated pH is used. In thiscase, the pH should be lowered upon the amplification reaction in orderto allow an oligonucleotide primer to bind to a target. A nucleic acidis successively amplified under isothermal conditions after denaturing adouble-stranded DNA into single-stranded DNAs or, if an RNA is used as atemplate, preparing a cDNA (a single-stranded DNA) by a reversetranscription reaction.

“Successively” means that a reaction proceeds without a change in thereaction temperature or the composition of the reaction mixture. As usedherein, “isothermal” means conditions of a substantially constanttemperature under which an enzyme and a nucleic acid strand function ineach step.

The nucleic acid amplification reaction of the present invention may beconducted at a normal temperature (e.g., 37° C.) by using a mesophilicDNA polymerase such as Klenow fragment. It can also be conducted at ahigh temperature (e.g., 50° C. or higher, or 60° C. or higher) by usingheat-resistant enzymes (an endonuclease and a DNA polymerase). In thiscase, non-specific annealing of a primer is suppressed, resulting inincrease in the specificity of DNA amplification. Furthermore, theproblem of forming secondary structure of a DNA as a template is solved,resulting in increase in the ability of extension of a DNA polymerase. Areverse transcription reaction and the nucleic acid amplification can besuccessively conducted in the method. In this case, a DNA having asequence derived from an RNA can be amplified by combining the use of areverse transcriptase with the above-mentioned reaction or by using aDNA polymerase having a reverse transcription activity.

In each aspect of the present invention, preferably, a chimericoligonucleotide primer that is complementary to a single-stranded DNA asa template is first annealed to the DNA, although it is not intended tolimit the present invention. A DNA that is complementary to the DNA asthe template (a primer-extended strand) is then extended along theremained sequence of the DNA as the template from the 3′-terminus of theprimer by the action of a DNA polymerase to synthesize a double-strandedDNA. An endonuclease acts on the double-stranded DNA and begins toreextend a DNA from the primer portion of the primer-extended strand denovo. In one aspect of the present invention, the endonuclease acts as anicking enzyme that introduces a nick into the double-stranded DNA or italters the structure of the double-stranded DNA composed of the chimericoligonucleotide primer and the DNA as the template, although the presentinvention is not restricted by a theory. A DNA polymerase having astrand displacement activity re-extends a DNA strand from the3′-terminus of the nick introduced in the double-stranded DNA togenerate a new primer-extended strand while releasing the DNA downstreamfrom the 3¹-terminus of the nick. Thus, the new primer-extended strandreplaces the previously synthesized primer-extended strand.

The method for amplifying a nucleic acid of the present invention can becarried out using two primers, i.e., a chimeric oligonucleotide primerthat is complementary to a nucleic acid as a template and anotherchimeric oligonucleotide primer that is complementary to a displacedstrand. In this case, one primer binds to a DNA strand as a template tocause a strand displacement reaction, whereas another primer binds to adisplaced strand released as a result of the strand displacementreaction to initiate another strand displacement reaction. It is clearthat a reaction product with one primer can function as a template foranother primer if this aspect is used. Thus, the amount of amplificationproduct increases in a non-linear manner as the amount of the templateincreases.

When the method for amplifying a nucleic acid of the present inventionis conducted using a double-stranded DNA as a template, both strands canserve as templates in the amplification reaction by using chimericoligonucleotide primers that anneal to the respective two strands. Ifthe reaction is initiated after denaturing the double-stranded DNA, achimeric oligonucleotide primer, four deoxyribonucleotide triphosphates(dNTPs), a DNA polymerase and an endonuclease are added to a reactionmixture before or after the double-stranded DNA is denatured. If heattreatment is used for denaturing the double-stranded DNA and aheat-resistant enzyme is not used, it is preferable to add the enzymeafter the denaturation.

In the aspect of the nucleic acid amplification method of the presentinvention in which a double-stranded DNA as a template and two chimericoligonucleotide primers are used, switching of templates may occur amongthe template-extended strand intermediates during the extension reactionfrom the primers to generate a double-stranded nucleic acid consistingof the synthesized primer-extended strands being annealed each other,although it depends on the reaction conditions or the like. Thedouble-stranded nucleic acid has chimeric oligonucleotide primers atboth ends. Then, reaction of extending complementary strands comprisingstrand displacement can be initiated from both of the ends again. As aresult of the reaction, an amplification product having the primersequence at one end is generated. Furthermore, if switching of templatesoccurs during the reaction, a double-stranded nucleic acid similar toone that described above is generated again.

The present invention provides a method for amplifying a nucleic acidwhich comprises using a DNA polymerase having a strand displacementactivity to effect a template switching reaction. In the templateswitching reaction in the presence of a double-stranded nucleoc acid asa template, two chimeric oligonucleotide primers substantiallycomplementary to the nucleotide sequences of the respective strands anda DNA polymerase having a strand displacement activity, twoprimer-extended strands complementary to the template are synthesized.Template switching of each of the primer-extended strands from thetemplate to the other primer-extended strand takes place during thesynthesis of the primer-extended strands.

As used herein; a template switching reation refers to a reaction inwhich when complementary strands are synthesized by strand displacementreactions from the both sides of a double-stranded nucleic acid, a DNApolymerase switches the template and synthesizes a complementary strandthereafter using the other complementary strand newly synthesized byanother DNA polymerase as a template. In other words, a templateswitching reaction refers to a reaction in which a double-strandednucleic acid as a template is treated with primers and a DNA polymerasehaving a strand displacement activity to generate extended strandscomplementary to the template, wherein a DNA polymerase that synthesizedthe primer-extended strands actively switches the template from theoriginal templates to the other primer-extended strands during thesynthesis of the extended strands. The ability of the DNA polymerase toeffect a template switching reaction can be determined, for example,according to the method as described in Example 45 below, although it isnot intended to limit the present invention.

A DNA polymerase capable of an effect the template switching reactionduring strand displacement reaction can be preferably used for thepresent invention. For example, a variant enzyme of Bca DNA polymeraselacking a 5′3′ exonuclease activity is preferably used in particular.Such an enzyme is commercially available as BcaBEST DNA polymerase(Takara Shuzo). It can also be prepared from Escherichia coliHB101/pU1205 (FERM BP-3720) which contains the gene for the enzymeaccording to the method as described in Japanese Patent No. 2978001.

Although it is not intended to limit the present invention, the mode ofreaction of the method for amplifying a nucleic acid of the presentinvention is considered as follows, for example.

In the method for amplifying a nucleic acid of the present invention, adouble-stranded nucleic acid as a template is treated in the presence ofan RNase H with two chimeric oligonucleotide primers that aresubstantially complementary to the nucleotide sequences of therespective strands and a DNA polymerase having a strand displacementactivity to synthesize primer-extended strands that are complementary tothe template. A double-stranded nucleic acid consisting of thesynthesized primer-extended strands being annealed each other and adouble-stranded nucleic acid consisting of the templates being annealedeach other to which the two primers are annealed as a result of templateswitching reaction can be obtained. The latter double-stranded nucleicacid is reused as a template.

The double-stranded nucleic acid consisting of the primer-extendedstrands being annealed each other is cleaved with an RNase H at sitesthat contain the ribonucleotides. Nucleic acids that are complementaryto the template are extended using a DNA polymerase having a stranddisplacement activity from the 3′-termini of the respective primerportions of the double-stranded nucleic acid by an effect of stranddisplacements. A double-stranded nucleic acid consisting of theprimer-extended strands being annealed each other and a double-strandednucleic acid consisting of the templates being annealed each other towhich the two primers are annealed as a result of template switchingreaction can be obtained.

If the template switching reaction does not take place, two types ofdouble-stranded nucleic acids each consisting of the template and theprimer-extended strand can be obtained.

Nucleic acids that are complementary to the template are extended usinga DNA polymerase having a strand displacement activity from the3′-termini of the respective primer portions of the double-strandednucleic acid. As a result of template switching reaction, adouble-stranded nucleic acid consisting of the primer-extended strandsbeing annealed each other and a double-stranded nucleic acid consistingof the templates being annealed each other to which the two primers areannealed can be obtained. The double-stranded nucleic acid to which thetwo primers are annealed is reused as a template.

If the template switching reaction does not take place, two types ofdouble-stranded nucleic acids each consisting of the template and theprimer-extended strand can be obtained.

The two types of double-stranded nucleic acids are cleaved with an RNaseH at sites that contain the ribonucleotides. Nucleic acids that arecomplementary to the template are extended using a DNA polymerase havinga strand displacement activity from the 3′-termini of the respectiveprimer portions of the double-stranded nucleic acid by an effect ofstrand displacements.

In the method for amplifying a nucleic acid of the present invention, adouble-stranded nucleic acid as a template is treated in the presence ofan RNase H with two chimeric oligonucleotide primers that aresubstantially complementary to the nucleotide sequences of therespective strands and a DNA polymerase having a strand displacementactivity and primer-extended strands that are complementary to thetemplate are synthesized. If the template switching reaction does nottake place, two types of double-stranded nucleic acids each consistingof the template and the primer-extended strand can be obtained.

In the amplification method of the present invention, the chimericoligonucleotide primer-extended strand may be cleaved at a site thatcontains the ribonucleotide such that the 5′ fragment (primer portion)resulting from the cleavage does not contain the ribonucleotide. Aprimer-extended strand extended from the thus generated primer portionis no longer cleaved by an endonuclease. As a result, an amplificationproduct having the primer sequence at its end is generated.

As described above, an amplification product without the primer sequenceand a product having the primer sequence(s) at one or both of the endsmay be generated in the nucleic acid amplification method of the presentinvention. These products are included in the amplification productsherein.

An example of the method for amplifying a nucleic acid of the presentinvention is illustrated in FIGS. 33 to 36. In other words, FIGS. 33 to36 illustrate an exemplary nucleic acid amplification in the nucleicacid amplification method of the present invention.

FIGS. 33 to 36 illustrate an exemplary nucleic acid amplification in thepresence of a DNA as a template which is a double-stranded nucleic acid,a pair of chimeric oligonucleotide primers synthesized on the basis ofthe nucleotide sequence information of the DNA as the template (in thefigures, the chimeric oligonucleotide primers have three ribonucleotidesat their 3′-termini; open circles in the figures represent theribonucleotides), a strand displacement type DNA synthetase (DNApolymerase) having a strand displacement activity, an RNase H which is aribonuclease that cleaves at a DNA-RNA hybrid site, and dNTPs which aresubstrates to be incorporated into the extended strands.

As shown in FIG. 33, the chimeric oligonucleotide primers synthesized onthe basis of the nucleotide sequence information of the DNA as thetemplate are annealed to the specific portions of the DNA as thetemplate. DNA strands are extended from the 3′-termini of the respectivechimeric oligonucleotide primers as a result of the strand displacementreaction as indicated with Step 1.

Next, as shown in FIG. 34, some of the primer-extended strands extendedfrom upstream and downstream are released from the original templates asa result of the template switching reaction as indicated with Step 2.The primer-extended strands are annealed each other at their 3′portions. Complementary strands are extended from the annealed extendedstrands, forming a double-stranded DNA consisting of the primer-extendedstrands being annealed each other. Additionally, a double-stranded DNAconsisting of the displaced strands being annealed each other to whichthe above-mentioned chimeric oligonucleotide primers are annealed isgenerated. This is utilized as the starting material in FIG. 34.

As indicated with Step 3 in FIG. 35, only one strand containing an RNAderived from the chimeric oligonucleotide primer of the double-strandedDNA in FIG. 34 consisting of the primer-extended strands being annealedeach other is cleaved by the action of an RNase H at the DNA/RNA hybridsite of the double-stranded DNA, resulting in introduction of a nick inthe double-stranded DNA.

Subsequently, the strand displacement reaction takes place from the nickin the double-stranded DNA and a DNA is extend as indicated with Step 4in FIG. 35. Next, a template switching reaction like that in Step 2 inFIG. 34 takes place in some degree or at some ratio as indicated withStep 5 in FIG. 35, resulting in a double-stranded DNA consisting of theamplification products, i.e., the primer-extended strands being annealedeach other.

In addition, a double-stranded DNA consisting of the displaced strandsbeing annealed each other to which the above-mentioned chimericoligonucleotide primers are annealed are generated.

Next, as shown in FIG. 36, DNA strands are extended as a result of thestrand displacement reaction from the 3′-termini of the respectivechimeric oligonucleotide primers in the double-stranded DNA in FIG. 35which consists of the displaced strands being annealed each other towhich the chimeric oligonucleotide primers are annealed. Similartemplate switching reactions take place in Step 2 and Step 5 in somedegree, resulting in a double-stranded DNA consisting of theprimer-extended strands being annealed each other. This double-strandedDNA is brought back to Step 3 in FIG. 35. The reaction starts on Step 3again. A double-stranded DNA which consists of the displaced strandsbeing annealed each other to which the chimeric oligonucleotide primersare annealed is generated and utilized as a starting material in FIG.36. As a result, a chain reaction, in which these double-strandednucleic acids are repeatedly generated, takes place to specificallyamplify and produce a region bounded by a pair of chimericoligonucleotide primers.

In the nucleic acid amplification method of the present invention usinga chimeric oligonucleotide, a polymer in which the regions to beamplified are connected each other may be generated. The polymer has astructure in which plural regions to be amplified are repeated in thesame direction. The polymers are observed upon electrophoretic analysisof amplification products as laddered bands. It is considered that thegeneration of such polymers is influenced by the region to be amplified,the size of the region, the flanking regions, the nucleotide sequence ofthe chimeric oligonucleotide primer to be used, the reaction conditionsand the like.

The polymer as described above contains plural regions to be amplified.For example, the polymer is useful when detection of a nucleic acidcontaining a region to be amplified is intended because it hybridizes toa number of probes upon hybridization using an appropriate probe andgenerates a intensive signal. The region to be amplified or a portionthereof can be obtained from the polymer as a monomer by using digestionwith a restriction enzyme or the like in combination.

The DNA polymerase used in the present invention should synthesize anextended strand from the 3′-terminus of the primer portion towards thedownstream while displacing a previously extended DNA strand. It isimportant that the DNA polymerase should not exhibit a 5′3′ exonucleaseactivity that may digest the displaced strand. For example, Klenowfragment, which is an exonuclease-deficient variant of DNA polymerase Ifrom Escherichia coli, a similar fragment derived from Bst DNApolymerase (New England Biolabs), and BcaBEST DNA polymerase from B. ca(Takara Shuzo) are useful as such a DNA polymerase. Sequenase 1.0 andSequenase 2.0 (United States Biochemical), and T5 DNA polymerase and φ29DNA polymerase as described in Gene, 97:13-19 (1991) can also be used. Apolymerase that normally has a 5′3′ exonuclease activity can be used inthe DNA synthesis method of the present invention if addition of anappropriate inhibitor can inhibit the activity.

The method for amplifying a nucleic acid of the present invention may beconducted at varying temperatures or it may be conducted isothermally.Varying temperatures means that the reaction temperatures are changedfor respective steps such that the change does not interfere with thereactions in the steps. Specifically, varying temperatures refers tochange in temperature to that suitable for, for example, each ofannealing of a primer, synthesis reaction of a complementary strand,nicking of a complementary strand and a displacement reaction.

On the other hand, isothermal means that the reaction temperature foreach step is unchanged and each step is conducted at a substantiallyconstant temperature. It is natural to select the temperature tooptimize the reaction conditions in both cases.

One feature of the method for amplifying a nucleic acid of the presentinvention is that the method does not require adjusting the temperatureup and down during the nucleic acid synthesis. Thus, the presentinvention provides a method for isothermally synthesizing a nucleicacid. Many of conventional nucleic acid amplification methods requireadjusting the temperature up and down to dissociate a target from asynthesized strand. These methods require a special reaction equipmentsuch as a thermal cycler for this purpose. However, the method of thepresent invention can be conducted only using an equipment that can keepa constant temperature. As described above, the method of the presentinvention can be conducted at a single temperature. Preferably, it isconducted by selecting the reaction temperature and the stringency levelsuch that non-specific annealing of a primer is reduced and such thatthe primer specifically anneals to a nucleic acid as a template.Although it is not intended to limit the present invention, the methodof the present invention can be conducted under high-temperatureconditions by using a heat-resistant enzyme as described above. Inaddition, it is preferable to conduct the method of the presentinvention at an appropriate temperature for sufficiently retaining theactivity of the enzyme used in order to maintain the reaction efficiencyat high level. Thus, the reaction temperature is preferably about 20° C.to about 80° C., more preferably about 30° C. to about 75° C., mostpreferably about 50° C. to about 70° C. although it varies depending onthe enzyme used. When the reaction is conducted under high-temperatureconditions in particular, it is preferable to use a longer primer thanthat for a reaction at a normal temperature. The sequence and the lengthof the primer appropriate for the reaction temperature may bedetermined, for example, with reference to its Tm value. Alternatively,a commercially available software for designing a primer such as OLIGO™Primer Analysis software (Takara Shuzo) may be used. For example, when areaction temperature of 55° C. to 60° C. or 65° C. is used, the primerused for the method of the present invention may be, for example,without limitation, 12-100 nucleotides in length, preferably 14-50nucleotides in length, more preferably 15-40 nucleotides in length. Anexample of effects brought by the elevated reaction temperature is thesolution of a problem of forming secondary structure of a DNA as atemplate. The elevated reaction temperature enables amplification of adesired nucleic acid even if a nucleic acid having a high GC content isused as a template. Furthermore, it is similarly effective in amplifyinga region of a long chain length. Such effect is observed in a rangebetween about 60 bp and about 20 kbp, specifically between about 110 bpand about 4.3 kbp, more specifically about 130 bp and about 1500 bp.

The amplification efficiency can be increased by adjusting the reactiontemperature in accordance with the GC content of the nucleic acid as thetemplate. For example, if a nucleic acid having a low GC content is usedas a template, the amplification reaction of the present invention canbe conducted at 50 to 55 C, although the temperature depends on thechain length to be amplified and the Tm value of the primer.

Use of a DNA polymerase having a reverse transcriptase activity (e.g.,BcaBEST DNA polymerase) in the method of the present invention can makethe amplification of a nucleic acid from an RNA, which comprises a stepof preparing a cDNA from an RNA (a reverse transcription reaction), beconveniently conducted. Alternatively, a product obtained byindependently conducting a step of preparing a cDNA from an RNA, i.e., acDNA, can be used in the method of the present invention as the DNA as atemplate.

In each case, the reaction in the method of the present invention isrepeated until it is terminated by appropriate means, for example, byinactivating the enzyme or by lowering the reaction temperature, oruntil the reaction is deprived of one of the substrates.

FIG. 40 illustrates one embodiment in which a single-stranded DNA as atemplate and two primers are used. Respective steps, which aresuccessively conducted in parallel, are described below:

-   -   (1) a step of annealing a single-stranded DNA as a template with        a chimeric oligonucleotide primer;    -   (2) a step of effecting a DNA extension reaction from the        3′-terminus of the primer to form a primer-extended strand;    -   (3) a step of cleaving at a site that contains a ribonucleotide        in the primer with an endonuclease;    -   (4) a step of effecting a strand displacement using a DNA        polymerase from the cleavage site in step (3);    -   (5) a step of reusing a double-stranded DNA, which is composed        of a template obtained in step (4) and a regenerated        primer-extended strand, in step (3), while utilizing a released        displaced strand in a reaction of step (6) and the following        steps;    -   (6) a step annealing an oligonucleotide primer that is different        from that in step (1) with the released displaced strand in        step (5) as a template;    -   (7) a step of effecting a DNA extension reaction from the        3′-terminus of the primer to form a primer-extended strand;    -   (8) a step of cleaving at a site that contains a ribonucleotide        in the primer with an endonuclease;    -   (9) a step of effecting a strand displacement using a DNA        polymerase from the cleavage site in step (8); and    -   (10) a step of reusing a template obtained in step (9) and a        regenerated primer-extended strand in step (8).

When a double-stranded DNA is used as a template, each of thesingle-stranded DNAs obtained after denaturing the double-stranded DNAserves as the template in step (1). Therefore, the amount ofamplification product is more than that obtained with a single-strandedDNA as a template. In addition, detection of the amplification productcan be conducted in a shorter time than that required when asingle-stranded DNA is used as a template.

The method for amplifying a nucleic acid of the present invention can beused for various experimental procedures that utilize amplification of anucleic acid including detection, labeling and sequencing of a nucleicacid.

Furthermore, the method for amplifying a nucleic acid of the presentinvention can be used for an in situ nucleic acid amplification method,a method for amplifying a nucleic acid on a solid substrate such as aDNA chip, or a multiplex nucleic acid amplification method in whichplural regions are simultaneously amplified.

One of the features of the method for amplifying a nucleic acid of thepresent invention is its ability to prepare a single-stranded DNA. Oneor two chimeric oligonucleotide primers can be used in the method forthis purpose. For example, if two oligonucleotide primers are used, themethod of the present invention can be conducted applying a similarprimer ratio to the so-called asymmetric-PCR in which an amplificationreaction is carried out by using an excess amount of one oligonucleotideprimer relative to another. The primer ratio is, without limitation,preferably in a range of 1:10 to 1:500, more preferably in a range of1:10 to 1:100. As a result, the amount of the replacement product fromone strand becomes excessive relative to that from another.

According to the method for amplifying a nucleic acid of the presentinvention, a single-stranded DNA substantially free of a complementarystrand thereto can be prepared. For example, a single-stranded DNA forproducing a material having an immobilized nucleic acid such as a DNAchip, a single-stranded DNA probe for detecting a target nucleic acid,or a mega-primer for the long-chain PCR can be readily produced in ashort time. Only a sense sequence or an antisense sequence can beselectively amplified by using the method of the present invention.Thus, the present invention is also useful as a method for producing anucleic acid having a sense sequence or a antisense sequence.

A region of a nucleic acid of interest can be amplified even from atrace amount of a nucleic acid as a template by conducting the method ofthe present invention in a buffer of Bicine, Tricine, HEPES, phosphateor tris.

Furthermore, the methods for amplifying a nucleic acid of the presentinvention does not require the specific reaction equipment that canadjust a temperature with the passage of time. Therefore, anamplification reaction can be conducted in a large volume of reactionmixture. Thus, a nucleic acid (e.g., for medical use) can beindustrially produced in large quantities.

The utilization efficiency of the primer in the method for amplifying anucleic acid of the present invention is about 100%, which may be 5- to10-fold higher than that in a conventional method such as the PCR.

The nucleic acid amplification method of the present invention canproduce an amplification product with high fidelity to the nucleotidesequence of the template nucleic acid. When the frequency of error inthe DNA synthesis in the method of the present invention was confirmedby analyzing the nucleotide sequences of resulting amplificationproducts, the frequency of error found in amplification productsobtained by the method of the present invention was equivalent to thatby LA-PCR which is known to be able to amplify a nucleic acid with highfidelity. In other words, the method of the present invention hasfidelity equivalent to that of the LA-PCR.

(6) Method for detecting target nucleic acid of the present inventionand kit for the method. A target nucleic acid in a sample can bedetected by using the method for amplifying a nucleic acid of thepresent invention. The detection method comprises:

-   -   (a) amplifying a target nucleic acid by the method for        amplifying a nucleic acid of the present invention as described        above; and    -   (b) detecting the target nucleic acid amplified in the step        above.

In step (a) above, if an RNA is used as a template, the reversetranscription reaction and the nucleic acid amplification reaction maybe conducted in one step. Although it is not intended to limit thepresent invention, for example, a combination of AMV RTase, MMLV RTaseor RAV-2 RTase and Bca DNA polymerase lacking 5′3′ exonuclease can bepreferably used as a combination of a reverse transcriptase and a stranddisplacement-type DNA polymerase.

The method can be utilized to detect or quantify a specific gene in asample. In other words, a specific gene can be detected or quantifiedfrom all samples suspected to contain a nucleic acid such as a DNA or anRNA. Examples of the samples from which a specific gene can be detectedor quantified include, but are not limited to, samples from organismssuch as a whole blood, a serum, a buffy coat, a urine, feces, acerebrospinal fluid, a seminal fluid, a saliva, a tissue (e.g., acancerous tissue or a lymph node) and a cell culture (e.g., a mammaliancell culture or a bacterial cell culture), samples that contain anucleic acid such as a viroid, a virus, a bacterium, a fungi, a yeast, aplant and an animal, samples suspected to be contaminated or infectedwith a micoorganism such as a virus or a bacterium (e.g., a food or abiological formulation), and samples that may contain an organism suchas a soil and a waste water. For example, a viroid, a virus, a fungi, abacterium or other microorganisms in a sample can be detected orquantified on the basis of the presence or the content of a specificgene derived from these microorganisms as a target. Particularly, amethod for detecting a pathogenic microorganism is useful in fields ofsanitation and environment. Furthermore, the method of the presentinvention can be used to distinguish a genotype of an organism or todetermine the expression level of a gene. Particularly, detection orconfirmation of the expression state of a disease-related gene, e.g., agene related to canceration of cells is useful in a field of medicine.Both of an RNA and a DNA can be preferably used as the nucleic acid asthe template in the detection.

The method for detecting a target nucleic acid of the present inventioncan be used to distinguish the difference in the nucleotide sequence ofthe target nucleic acid. In this aspect, the chimeric oligonucleotideprimer to be used is designed such that the 3′-terminal portion of theprimer is positioned close to the specific base of the target nucleotidesequence to be distinguished. For example, it is designed such that ahydrogen bond is formed between the base and the 3′-terminal base of theprimer. If a mismatch exists between the nucleotide sequence of the3′-terminal portion of the primer and the nucleotide sequence of thetemplate, amplification from the target nucleic acid does not take placeand no amplification product is generated using the above-mentionedchimeric oligonucleotide primer for amplification reaction. Informationconcerning a specific base in a gene such as a point mutation or asingle nucleotide polymorphism (SNP) can be obtained using the method.

The method for detecting a target nucleic acid of the present inventioncan be conducted by amplifying the target nucleic acid directly from asample containing the nucleic acid. In this case, the chain length ofthe target nucleic acid to be amplified is not limited to a specificone. For example, a region of 200 bp or shorter, preferably 150 bp orshorter is effective for sensitive detection of the target nucleic acid.The target nucleic acid in the sample can be detected with highsensitivity by designing the chimeric oligonucleotide primers of thepresent invention to result in the chain length to be amplified asdescribed above.

In addition, a target nucleic acid can be detected with more sensitivityeven from a trace amount of a nucleic acid sample in the detectionmethod of the present invention by using a reaction buffer containingBicine, Tricine, HEPES, phosphate or tris as a buffering component andan annealing solution containing spermidine or propylenediamine asexemplified in (4) above. In this case, the endonuclease and the DNApolymerase to be used are not limited to specific ones. For example, acombination of an RNase H from Escherichia coli, a bacterium of genusPyrococcus or a bacterium of genus Archaeoglobus and BcaBEST DNApolymerase is preferable. It is considered that the preferable units ofthe endonuclease and the DNA polymerase may vary depending on the typesthe enzymes. In such a case, the composition of the buffer and theamount of the enzymes added may be adjusted using the increase indetection sensitivity or the amount of amplification product as anindex.

In the detection method of the present invention, dUTP may beincorporated as a substrate during amplification of a target nucleicacid. Thus, if dUTP is used as a substrate, it is possible to preventcarry-over contamination of amplification products by degradingamplification products utilizing uracil N-glycosidase (UNG).

Known methods for detecting a nucleic acid can be used for step (b).Examples of such methods include detection of a reaction product havinga specific size by electrophoresis, and detection by hybridization witha probe. Furthermore, a detection method in which magnetic beads arecombined can be preferably used. A fluorescent substance such asethidium bromide is usually used in the detection by electrophoresis.The hybridization with a probe may be combined with the detection byelectrophoresis. The probe may be labeled with a radioisotope or with anon-radioactive substance such as biotin or a fluorescent substance.Additionally, use of a labeled nucleotide in step (a) may facilitate thedetection of amplification product into which the labeled nucleotide isincorporated, or may enhance the signal for detection utilizing thelabel. Furthermore, a fluorescence polarization method, a fluorescenceenergy transition or the like can also be utilized for the detection.The target nucleic acid can be detected automatically or quantified byconstructing a suitable detection system. In addition, detection withnaked eyes by a hybrid chromatography method can be preferably used.

A ribonucleotide (RNA) probe labeled with two or more fluorescentsubstances positioned at a distance that results in a quenching statecan be used in the detection method of the present invention. The probedoes not emit fluorescence. When it is annealed to a DNA amplified froma target nucleic acid that is complementary to the probe, RNase Hdigests the probe. The distance between the fluorescent substances onthe probe then increases, resulting in the emission of fluorescence.Thus, the emission reveals the presence of the target nucleic acid. IfRNase H is used in the method for amplifying a nucleic acid of thepresent invention, a target nucleic acid can be detected only by addingthe probe to the reaction mixture. For example, a combination offluorescent substances, 6-carboxyfluorescein (6-FAM) andN,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), can be preferably usedfor labeling the probe.

The present invention further provides a probe used in theabove-mentioned method for detecting a target nucleic acid. The probe ofthe present invention is not limited to specific one as long as it canhybridize to a target nucleic acid amplified by the nucleic acidamplification method of the present invention under normal hybridizationconditions. In view of specific detection of amplification product, aprobe that hybridizes under conditions, for example, known to thoseskilled in the art as being stringent is preferable. The stringenthybridization conditons are described in, for example, T. Maniatis etal. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., 1989, ColdSpring Harbor Laboratory. Specifically, the stringent conditions areexemplified by the following: incubation at a temperature about 25 Clower than the Tm of the probe to be used for 4 hours to overnight in6×SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing0.5% SDS, 5× Denhardt's (0.1% bovine serum albumin (BSA), 0.1%polyvinylpyrrolidone, 0.1% Ficoll 400) and 100 mg/ml salmon sperm DNA. Aprobe having a label as described above may be used as the probe forfacilitating the detection of the target nucleic acid.

The method for amplifying a nucleic acid under isothermal conditions ofthe present invention does not require the use of an equipment such as athermal cycler. The number of primers used in the amplification methodof the present invention can be one or two, which is less than that usedin a conventional method. Since reagents such as dNTPs used for PCR andthe like can be applied to the method of the present invention, therunning cost can be reduced as compared with a conventional method.Therefore, the method of the present invention can be preferably used,for example, in a field of genetic test in which the detection isroutinely conducted. The method of the present invention provides agreater amount of an amplification product in a shorter time than thePCR. Therefore, the method of the present invention can be utilized as aconvenient, rapid and sensitive method for detecting a gene.

In genetic analysis on a genomic level, attempts are made to make thereaction system small and to increase the degree of integration in orderto analyze a large amount of nucleotide sequences. An system has beendeveloped for this purpose by utilizing the latest hyperfine processingtechniques in which basic processes for genome analysis (e.g.,extraction of DNA from cells, DNA amplification reaction,electrophoresis, hybridization, detection of the DNA of interest, etc.)are integrated on a microchip of several square centimeters to fingertipsize. Such a system is called as a microchip or a nanochip.

Application of the PCR to the system as a gene amplification reaction iscurrently considered. However, since the PCRs requires means forrepeatedly controlling temperature up and down over time, the systemwould become complicated. By contrast, since the system can besimplified using the method of the present invention which can amplify anucleic acid under isothermal conditions, the method is quite suitablyutilized for the integrated system as described above. A highlyintegrated system can be constructed by utilizing the techniques of thepresent invention.

(7) Kit of the Present Invention

The present invention provides a kit used for the method for amplifyingor detecting a nucleic acid of present invention as described above. Inone embodiment, the kit is in a packaged form and contains instructionsregarding the use of a DNA polymerase and an endonuclease in a stranddisplacement reaction. Also, a kit that contains a DNA polymerase havinga strand displacement activity, an endonuclease, and a buffer for astrand displacement reaction is preferably used for the method of thepresent invention. Alternatively, a commercially available DNApolymerase having a strand displacement activity and/or endonuclease maybe selected and used according to the instructions. Additionally, thekit may contain a reagent for a reverse transcription reaction that isused when an RNA is used as a template. The DNA polymerase can beselected from the DNA polymerases to be used in the present invention asdescribed above in (3). The endonuclease can be selected from theendonucleases as described above in (2). One having the reaction buffercomposition as described above in (4) can be preferably used as thebuffer for the strand displacement reaction.

“Instructions” are printed matters describing a method of using the kit,e.g., a method for preparing a reagent solution for a stranddisplacement reaction, recommended reaction conditions and the like. Theinstructions include an instruction manual in a form of a pamphlet or aleaflet, a label stuck to the kit, and description on the surface of thepackage containing the kit. The instructions also include informationdisclosed or provided through electronic media such as the Internet.

The kit of the present invention may further contain a reaction buffercontaining Bicine, Tricine, HEPES, phosphate or tris as a bufferingcomponent and an annealing solution as exemplified in (4) above.Additionally, it may contain a DNA polymerase having a stranddisplacement activity and an RNase H. Furthermore, the kit may contain amodified deoxyribonucleotide or a deoxynucleotide triphosphate analog.

The kit used in the method for detecting a target nucleic acid mayfurther contain an appropriate chimeric oligonucleotide primer foramplifying the target nucleic acid and a reagent for detecting theamplified target nucleic acid such as a probe in addition to theinstructions and the reagents for amplification reaction as describedabove.

In addition, the kits of the present invention include a kit containingthe chimeric oligonucleotide primer used in the present invention and/orthe probe of the present invention as described above.

(8) Composition of the Present Invention.

The present invention provides a composition used for the method foramplifying a nucleic acid of the present invention or the method fordetecting a nucleic acid of the present invention as described above.For example, the composition may contain the endonuclease as describedin (2) above and the DNA polymerase as described in (3) above. Thecomposition may further contain a buffering component, a magnesium salt,dNTPs and the like as components for conducting an amplificationreaction. Furthermore, it may contain a modified deoxyribonucleotide ora deoxynucleotide triphosphate analog. Those as described in (4) abovecan be used as the buffering component and other additives.

In a particularly preferable aspect, the composition may containsuitable amounts of the various components as listed above for thenucleic acid amplification method of the present invention.Amplification reaction can be conducted only by adding an appropriatetemplate and chimeric oligonucleotide primer(s) to the composition. Ifthe object to be amplified is known beforehand, the compositionpreferably contains chimeric oligonucleotide primer(s) suitable foramplifying the object.

(9) Material Having Immobilized Nucleic Acid Arrayed in PredefinedRegion of the Present Invention and Method for Producing the Same.

A DNA chip (also referred to as a DNA microarray or a DNA array) is amaterial having an immobilized nucleic acid in which various fragmentsof genes or DNAs are arrayed and immobilized in a predefined region orat a predefined position on a solid substrate such as a slide glass. TheDNA chip is used for examining the presence of a nucleic acid in anucleic acid sample that has a sequence complementary to an arrayed andimmobilized DNA in a predefined region on the DNA chip. The examinationis carried out by contacting the DNA chip with the nucleic acid sampleprepared from a sample, preferably a labeled nucleic acid sample, forhybridization. Since the DNA chip can be used to detect or quantify anumber of nucleic acids in a sample in one procedure, it is a veryuseful means that dramatically promotes the analysis of gene expression,or the analysis of a mutation or polymorphism. A DNA chip in which adouble-stranded nucleic acid is arrayed and immobilized in a predefinedregion is used for hybridization after it is subjected to appropriatedenaturation. A DNA chip in which a single-stranded DNA complementary toa target nucleic acid to be detected is arrayed and immobilized in apredefined region is particularly preferable for the detection of atarget nucleic acid.

As described above, a desired DNA can be amplified in a single-strandedform by the method of the present invention. Although any method forpurifying an amplification product can be used, purification usingisopropanol precipitation is preferable. The thus obtained DNA,preferably a single-stranded DNA substantially free of a complementarystrand thereto, can be preferably used as a DNA fragment to beimmobilized onto a DNA chip. Thus, the method of the present inventionis preferably used as a method for preparing a DNA to be arrayed andimmobilized in a predefined region for producing a DNA chip. Anyinsoluble substrate can be used as a substrate onto which the thusobtained DNA is arrayed and immobilized in a predefined region, but aplate-shaped substrate made from glass or plastic, and a membrane-shapedsubstrate made from nitrocellulose or nylon are preferably used. A knownmethod for immobilizing a nucleic acid can be used for theimmobilization. The DNA may be directly immobilized onto a substrate.Alternatively, the DNA may be immobilized through a suitable linker orafter ligating plural DNA molecules. Furthermore, since a modifieddeoxyribonucleotide can be incorporated into an amplified nucleic acidby the method of the present invention, the material having animmobilized nucleic acid can be produced utilizing the modificationgroup.

A target nucleic acid that hybridizes with a nucleic acid on a materialhaving an immobilized nucleic acid in which a DNA amplified by themethod of the present invention is arrayed and immobilized in apredefined region (e.g., a DNA chip) can be detected or quantified. Suchdetection or quantification can be accomplished by contacting thematerial with a nucleic acid sample prepared from a sample suspected tocontain the target nucleic acid for hybridization. Among these, a DNAchip in which a single-stranded DNA amplified by the method of thepresent invention is arrayed and immobilized in a predefined regionallows the detection of a target nucleic acid with more convenientoperation, higher sensitivity and higher reproducibility as comparedwith a conventional material.

(10) Method for Producing Nucleic Acid in large Quantities of thePresent Invention.

As described above, one aspect of the present invention provides amethod for amplifying a nucleic acid that can be carried out underisothermal conditions. A desired nucleic acid can be produced in themethod by mixing a nucleic acid as a template for the nucleic acid to beamplified and various components required for a reaction and reactingthe mixture under isothermal conditions. Since the PCR requires changingthe temperature of the reaction mixture over time, the reaction volumeis limited to one in which the temperature can be controlled (usually,200 μl or less). Therefore, it is difficult to scale up the volume. Onthe other hand, there is no such limitation in the method of the presentinvention. A large amount of nucleic acid can be produced by increasingthe volume of the reaction mixture. In the method of the presentinvention, the volume is, for example, preferably more than 200 μl, morepreferably 300 μl or more, and most preferably 500 μl or more, althoughit is not intended to limit the present invention. In the method of thepresent invention, a number of complementary strand molecules aresynthesized from one template molecule. Furthermore, nucleic acids canbe synthesized using these complementary strand molecules as templates.Thus, a desired nucleic acid can be efficiently produced in largequantities by suitably selecting the template and the primer.Additionally, the fact that, unlike the PCR, the method of the presentinvention does not require a special equipment or a complicatedtemperature change makes it advantageous in view of the cost ofequipment and energy. Therefore, the method is an excellent industrialmethod for producing a nucleic acid in large quantities.

The nucleic acid of interest can be amplified or produced even from atrace amount of a nucleic acid as a template in the production method ofthe present invention by using the reaction buffer and the annealingsolution as exemplified in (4) above. In this case, the endonuclease andthe DNA polymerase to be used are not limited to specific ones. Forexample, a combination of an RNase H from Escherichia coli and BcaBESTDNA polymerase is preferable. It is considered that the preferable unitsof the endonuclease and the DNA polymerase may vary depending on thetypes of the enzymes. In such a case, the composition of the buffer andthe amount of the enzymes added may be adjusted using the maximal amountof amplification product as an index.

Furthermore, the method of the present invention is useful as a methodfor supplying a variety of DNA fragments in large quantities, such asthose to be immobilized onto the DNA chip. Specifically, DNA fragmentscan be obtained in large quantities in simple reaction steps in oneembodiment. In another embodiment, a limited number of primers can beused to obtain a variety of DNA fragments. A step of amplifying thenucleic acid that serves as the template in the method of the presentinvention beforehand by a known nucleic acid amplification method suchas the PCR can be incorporated in the latter embodiment. All kinds ofnucleic acids as templates can be amplified using a limited number ofprimers, for example, based on the method for amplifying a nucleic acidusing a random primer having a tag sequence (Nucleic Acids Research,24(19):3778-3783 (1996)) or the degenerate oligonucleotide-primed PCR(DOP-PCR; Genomics, 13:718-725 (1992)), which uses a degenerate primer.The nucleic acid amplification method of the present invention can beconducted using one or several primers for all of the nucleic acids astemplates produced in the above-mentioned step. This can be accomplishedby designing the primer used in the nucleic acid amplification method ofthe present invention such that it corresponds to the tag sequence addedto the random or degenerate primer. Thus, a combination of a suitablestep for preparing a nucleic acid as a template and the method of thepresent invention can supply a variety of DNA fragments in largerquantities and at a lower cost as compared with a conventional method.

A pharmaceutical composition containing a nucleic acid may contain adouble-stranded DNA for expressing a useful polypeptide in a cell or asingle-stranded antisense DNA for suppressing the expression of a geneof interest. Such a nucleic acid is administered into an organism usingsuitable means, for example, a carrier for gene transfer such asliposome. The method for producing a nucleic acid of the presentinvention is preferable for producing a single-stranded ordouble-stranded nucleic acid for medical use in large quantities.Additionally, a nucleic acid containing a DNTP analog that, for example,suppresses the degradation of the nucleic acid in vivo can be readilyproduced by the method of the present invention.

Since the DNA fragment amplified in the present invention is composed ofnormal nucleotides, the amplified DNA can be, for example, subclonedinto a suitable vector utilizing a restriction enzyme site in the DNA.Furthermore, the DNA can be treated with a restriction enzyme for RFLPwithout a problem, for example. Therefore, the DNA can be widelyutilized in a field of genetic test. In addition, a promoter sequencefor an RNA polymerase can be incorporated into the amplified fragment.The amplified fragment can be used as a template to synthesize an RNA,which can be used as a probe, for example. Of course, afluorescence-labeled DNA probe can be produced by conducting the methodfor amplifying a nucleic acid of the present invention using afluorescence-labeled DNTP instead of a normal DNTP.

Features of the method for amplifying a nucleic acid of the presentinvention are listed below.

1. It can amplify a large amount of a nucleic acid from a small amountof a template. The amplification product increases quadratically whentwo primers are used.

2. It can be conducted isothermally. In this case, it does not requirethe use of an equipment such as a thermal cycler. Therefore, thereaction volume can be readily scaled up.

3. Usually, the amplification reaction is conducted using one or twochimeric oligonucleotide primer and two enzymes (a DNA polymerase and anendonuclease).

4. Since a number of DNA strands are synthesized from one molecule of aprimer, the amount of the primer does not restrict the amount of theamplification product. Furthermore, the primer utilization efficiency isabout 100%, which is very higher than that of the PCR.

5. A single-stranded or double-stranded DNA can be selectively amplifieddepending on the purpose.

6. Since it does not require a DNTP analog such as an (α-S) DNTP for theamplification reaction, the cost of reagents is low. Furthermore, anucleic acid in a natural form without a dNTP analog can be obtained.

7. It can supply an amplified DNA fragment at low cost and in largequantities by combining the method of the present invention with anothernucleic acid duplication method.

8. The detection method of the present invention has an equal or higherdetection sensitivity as compared with that of a conventional method.The detection method of the present invention can be conducted in ashorter time than that required for a conventional method having theequal sensitivity.

9. The method is suitable for amplification of a nucleic acid, automateddetection, detection in a small amount and highly integrated detectionon a microchip or a nanochip.

As described above, the method of the present invention is suitable fordetection of a gene and industrial production of a nucleic acid.

EXAMPLES

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Referential Example 1 Preparation of RNase H from Thermophile BacillusCaldotenax

Bacillus caldotenax YT-G (purchased from Deutsche Sammlung vonMikroorganismen; DSM406) was inoculated into 100 ml of a mediumcontaining 0.2% Tryptone (Difco Laboratories) and 1.5% yeast extract(Difco Laboratories) (pH 6.5), cultured at 60 C for 140 minutes withshaking and used as a pre-culture. 30 ml of the pre-culture wasinoculated into 3 L of a medium having the same composition and culturedwith aeration at 2.5 L/minute and stirring at 250 rpm at a temperatureof 60 C for 5 hours.

The cells were collected by centrifuging the culture at 5000×g for 15minutes. 402 g (wet weight) of the cells were suspended in 1000 ml of 50mM tris-HCl buffer (pH 7.5) containing 10 mM mercaptoethanol, 0.5 MNaCl, 1 mM EDTA and 20 μM PAPMSF and disrupted using MINI-Lab (APVGAULIN/RANNIE). Cell debris were removed by centrifugation to recover asupernatant.

A polyethylene imine solution was added to the resulting supernatant ata final concentration of 0.1%. After stirring, the mixture was allowedto stand for 1 hour. A supernatant was then recovered by centrifugation.Ammonium sulfate was added to the supernatant to 50% saturation. Aprecipitate obtained by centrifugation was dissolved in 20 mM tris-HClbuffer (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mM EDTA, 50 mMNaCl and 10% glycerol. The solution was dialyzed against the samebuffer. The dialyzed sample was loaded onto a 280-ml DE52 column(Whatman) equilibrated with the same buffer and non-adsorptive fractionswere collected.

The column was further washed with 420 ml of the buffer used for theequilibration and washing fractions were collected. The non-adsorptivefractions and the washing fractions from the DE52 column chromatographywere mixed together and loaded onto a 240-ml P-11 column (Whatman)equilibrated with 20 mM tris-HCl buffer (pH 7.5) containing 10 mMmercaptoethanol, 0.1 mM EDTA, 50 mM NaCl and 10% glycerol. Elution wasthen carried out using the equilibration buffer containing 0 to 0.5 MNaCl.

The resulting active fractions were placed in a dialysis tube. The tubewas placed on solid polyethylene glycol 20000 fordehydration-concentration at 4 C. The enzyme concentrate was then loadedonto a 300-ml Superdex G-200 column (Amersham Pharmacia Biotech)equilibrated with 25 mM tris-HCl buffer (pH 7.5) containing 5 mMmercaptoethanol, 0.5 mM EDTA, 30 mM NaCl and 50% glycerol. Elution wascarried out using the buffer used for equilibration to obtain activefractions. The active fractions were loaded onto a 15-mlHeparin-Sepharose column (Amersham Pharmacia Biotech) equilibrated with20 mM tris-HCl buffer (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mMEDTA, 50 mM NaCl and 10% glycerol. Elution was carried out using theequilibration buffer containing 0 to 0.5 M NaCl.

The resulting active fractions were loaded onto a 5-ml Hitrap-SP column(Amersham Pharmacia Biotech) equilibrated with 20 mM tris-HCl buffer (pH7.5) containing 10 mM mercaptoethanol, 0.1 mM EDTA, 50 mM NaCl and 10%glycerol. Elution was carried out using the equilibration buffercontaining 0 to 0.5 M NaCl. The resulting active fractions were loadedonto a 300-ml Superdex G-200 column (Amersham Pharmacia Biotech)equilibrated with 25 mM tris-HCl buffer (pH 7.5) containing 5 mMmercaptoethanol, 0.5 mM EDTA, 30 mM NaCl and 50% glycerol again. Theresulting active fractions were used as an RNase H preparation (anenzyme solution).

A heat-resistant RNase H activity was measured as follows.

1 mg of poly(rA) or poly(dT) (both from Amersham Pharmacia Biotech) wasdissolved in 1 ml of 40 mM tris-HCl (pH 7.7) containing 1 mM EDTA toprepare a poly(rA) solution and a poly(dT) solution.

The poly(rA) solution (to a final concentration of 20 μg/ml) and thepoly(dT) solution (to a final concentration of 30 μg/ml) were then addedto 40 mM tris-HCl (pH 7.7) containing 4 mM MgCl₂, 1 mM DTT, 0.003% BSAand 4% glycerol. The mixture was reacted at 37 C for 10 minutes and thencooled to 4 C at prepare a poly(rA)-poly(dT) solution.

1 μl of an enzyme solution was added to 100 μl of the poly(rA)-poly(dT)solution. The mixture was reacted at 40 C for 10 minutes. 10 μl of 0.5 MEDTA was added thereto to terminate the reaction. Absorbance at 260 nmwas then measured. As a control, 10 μl of 0.5 M EDTA was added to thereaction mixture, the resulting mixture was reacted at 40 C for 10minutes, and the absorbance was then measured. A value (difference inabsorbance) was obtained by subtracting the absorbance for the controlfrom the absorbance for the reaction in the absence of EDTA. Thus, theconcentration of nucleotide released from poly(rA)-poly(dT) hybrid bythe enzymatic reaction was determined on the basis of the difference inabsorbance. One unit of an RNase H was defined as an amount of enzymethat increases A₂₆₀ corresponding to release of 1 nmol of ribonucleotidein 10 minutes calculated according to the following equation. If adiluted enzyme solution is used, the value obtained using the followingequation was corrected based on the dilution rate:Unit=[Difference in Absorbance×Reaction Volume(ml)]/0.0152

Referential Example 2 Cloning of Bacillus Caldotenax RNase HII Gene

(1) Preparation of Genomic DNA from Bacillus Caldotenax

Bacillus caldotenax YT-G (DSM406) was inoculated into 60 ml of LB medium(1% Tryptone, 0.5% yeast extract and 0.5% NaCl, pH 7.2) and cultured at65 C for 20 hours. After culturing, the culture was centrifuged tocollect cells. The cells were suspended in 2 ml of 25% sucrose and 50 mMtris-HCl (pH 8.0). 0.2 ml of 10 mg/ml lysozyme chloride (Nacalai Tesque)in water was added thereto. The mixture was reacted at 20 C for 1 hour.After reaction, 12 ml of a mixture containing 150 mM NaCl, 1 mM EDTA and20 mM tris-HCl (pH 8.0), 0.1 ml of 20 mg/ml proteinase K (Takara Shuzo)and 1 ml of a 10% aqueous solution of sodium lauryl sulfate were addedto the reaction mixture. The mixture was incubated at 37 C for 1 hour.

2.1 ml of 5 M NaCl and 2 ml of a CTAB-NaCl solution [10%cetyltrimethylammonium bromide (Nacalai Tesque) and 0.7 M NaCl] werethen added to the mixture and the resulting mixture was mixed thoroughlyand incubated at 65 C for 10 minutes. An equal volume of a mixture ofchloroform/isoamyl alcohol (24:1, v/v) was added thereto. The resultingmixture was gently mixed for 10 minutes and then centrifuged for 10minutes at 10000×g. After centrifugation, an equal volume of a mixtureof phenol saturated with 100 mM tris-HCl (pH 8.0)/chloroform/isoamylalcohol (25:24:1, v/v) was added to the resulting supernatant. Theresulting mixture was gently mixed for 10 minutes and then centrifugedfor 10 minutes at 10000×g. After centrifugation, 0.6 volume of2-propanol was added to the resulting supernatant. The resulting fibrousprecipitate was wound using a glass bar, washed with 70% ethanol inwater, air-dried and then dissolved in 0.5 ml of TE buffer to obtain agenomic DNA solution.

(2) Cloning of a Middle Portion of RNase HII Gene

Oligonucleotides BsuII-3 and BsuII-6 represented by SEQ ID NOS:97 and 98were synthesized on the basis of Motif I and Motif III, portionsconserved among amino acid sequences of RNase HIIs from variousorganisms (Biochemistry, 38:605-608 (1999)).

A PCR was carried out in a volume of 100 μl using 1 μl of the Bacilluscaldotenax genomic DNA solution as prepared in Referential Example 2-(1)as a template, and 100 pmol of BsuII-3 and 100 pmol of BsuII-6 asprimers. TaKaRa Taq polymerase (Takara Shuzo) was used as a DNApolymerase for the PCR according to the attached protocol. The PCR wascarried out as follows: 50 cycles of 94 C for 30 seconds, 45 C for 30seconds and 72 C for 1 minute. After reaction, the reaction mixture wassubjected to phenol treatment followed by ethanol precipitation topurify a DNA. The resulting DNA was blunt-ended using T4 DNA polymerase(Takara Shuzo) and then subjected to agarose gel electrophoresis torecover an amplified DNA fragment of about 0.4 kb from the gel. Theabout 0.4-kb DNA fragment was ligated with pUC119 (Takara Shuzo)digested with Sma I (Takara Shuzo) using T4 DNA ligase (Takara Shuzo).The ligation mixture was used to transform Escherichia coli JM109. Theresulting transformants were cultured to obtain a plasmid 21-12 intowhich the about 0.4-kb DNA was inserted.

(3) Cloning of Upstream Portion of RNase II Gene

The nucleotide sequence of the inserted fragment of about 0.4 kb in theplasmid 21-12 obtained in Referential Example 2-(2) was determined.Oligonucleotides RNII-S1 and RNII-S2 represented by SEQ ID NOS:99 and100 were synthesized on the basis of the determined nucleotide sequence.

The Bacillus caldotenax genomic DNA as prepared in Referential Example2-(1) was digested with BamHI (Takara Shuzo) and ligated with a Sau3AIcassette (Takara Shuzo) using T4 DNA ligase. A procedure was carried outaccording to the protocol attached to TaKaRa LA PCR in vitro cloning kit(Takara Shuzo) using the ligation mixture as a template, RNII-S2 as aprimer for a primary PCR and RNII-S1 as a primer for a secondary PCR. ADNA was purified from the solution after the secondary PCR by phenolextraction followed by ethanol precipitation. The DNA was blunt-endedusing T4 DNA polymerase and then subjected to agarose gelelectrophoresis to recover an amplified DNA fragment of about 1.5 kbfrom the gel. The about 1.5-kb DNA fragment was ligated with pUC119digested with Sma I using T4 DNA ligase. The ligation mixture was usedto transform Escherichia coli JM109.

The resulting transformants were cultured to obtain a plasmid B25N16into which the about 1.5-kb DNA was inserted.

(4) Cloning of the Entire RNase II Gene

Oligonucleotides RNII-S5 and RNII-S6 represented by SEQ ID NOS:101 and102 were synthesized on the basis of the nucleotide sequence of theinserted fragment of about 0.4 kb in the plasmid 21-12 as determined inReferential Example 2-(3).

A PCR was carried out using the plasmid 21-12 as prepared in ReferentialExample 2-(2) as a template, and RNII-S5 and RNII-S6 as primers. TaKaRaEx Taq polymerase (Takara Shuzo) was used as a DNA polymerase for thePCR according to the attached protocol. The PCR was carried out asfollows: 25 cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 Cfor 30 seconds. After reaction, the reaction mixture was subjected toagarose gel electrophoresis. An amplified DNA fragment of about 0.3 kbwas recovered from the gel. The about 0.3-kb DNA fragment was labeledwith digoxigenin using DIG High-prime (Roche Diagnostics).

Southern Hybridization was carried out for digests of the Bacilluscaldotenax genomic DNA as prepared in Referential Example 2-(1) withHindIII (Takara Shuzo), Sac I (Takara Shuzo), or HindIII and SacI usingthe digoxigenin-labeled DNA as a probe. Hybridization and detection werecarried out using DIG Luminescent Detection Kit (Roche Diagnostics)according to the protocol attached thereto. As a result, DNA fragmentsof about 4.5 kb, about 5.8 kb and about 1.3 kb were hybridized with theprobe for the digests with HindIII, SacI, and HindIII and SacI,respectively.

Based on these results, the Bacillus caldotenax genomic DNA was digestedwith HindIII and subjected to agarose gel electrophoresis to recoverDNAs of about 4.5 kb from the gel. The resulting DNA fragments weredigested with SacI and subjected to agarose gel electrophoresis torecover DNAs of about 1.3 kb from the gel. The resulting DNAs wereligated with pUC19 (Takara Shuzo) digested with HindIII and SacI usingT4 DNA ligase. The ligation mixture was used to transform Escherichiacoli HB101.

The resulting transformants were replica-plated onto Hybond-N™ (AmershamPharmacia Biotech). Colony hybridization was then carried out using theabove-mentioned digoxigenin-labeled probe according to a conventionalmethod. A plasmid pRHB1 was prepared from the thus obtained positiveclone.

The nucleotide sequence of the DNA inserted into pRHB1 was thendetermined. Comparison of the amino acid sequence deduced from thenucleotide sequence with the amino acid sequence of the RNase HII fromBacillus subtilis suggested that a region of about 40 bp from theinitiation codon was missing in the DNA in pRHB1. Then, the full-lengthRNase H gene was constructed as follows.

B25N16 as prepared in Referential Example 2-(3) was digested withHindIII and subjected to agarose gel electrophoresis to recover a DNAfragment of about 160 bp from the gel. The about 160-bp DNA fragment wasligated with pRHB1 digested with HindIII using T4 DNA ligase. Theligation mixture was used to transform Escherichia coli HB101. Plasmidswere prepared from the resulting transformants.

Next, an oligonucleotide RNII-Nde represented by

SEQ ID NO:103 was synthesized on the basis of the presumed nucleotidesequence around the initiation codon. PCRs were carried out using theplasmids prepared from the transformants as templates, and RNII-Nde andRNII-S6 as primers. A plasmid from which a DNA fragment of about 0.7 kbwas amplified was selected and designated as pRHB11.

The nucleotide sequence of the DNA fragment inserted into the thusobtained plasmid pRHB11 was determined. Analysis of the results revealedan open reading frame presumably encoding RNase HII. This nucleotidesequence is shown in SEQ ID NO:104. The amino acid sequence of RNase HIIdeduced from the nucleotide sequence is shown in SEQ ID NO:105.

Escherichia coli HB101 transformed with the plasmid pRHB11 is designatedand indicated as Escherichia coli JM109/pRHB11, and deposited on Sep. 5,2000 at the International Patent Organism Depositary, National Instituteof Advanced Industrial Science and Technology under accession numberFERM P-18021.

(5) Expression of Bacillus Caldotenax RNase HII Gene

Escherichia coli HB101 transformed with pRHB11 or pRHB1 was inoculatedinto 5 ml of LB medium containing 100 μg/ml of ampicillin and culturedwith shaking at 37 C overnight. After cultivation, cells collected bycentrifugation were suspended in 0.5 ml of TE buffer and sonicated. Asupernatant obtained by centrifugation was used as a cell crude extract.

10 mM tris-HCl (pH 8.0), 1 mM dithiothreitol (Nacalai Tesque), 0.003%bovine serum albumin (fraction V, Sigma), 4% glycerol, 20 μg/ml poly(dT)(Amersham Pharmacia Biotech) and 30 μg/ml poly(rA) (Amersham PharmaciaBiotech) were mixed together. The mixture was incubated at 37 C for 10minutes and used as a substrate solution for measuring an RNase Hactivity.

1 μl of 1 M MnCl₂ was added to 100 μl of the substrate solution. Themixture was incubated at 40 C. 10 μl of a 10-fold dilution of the cellcrude extract was added to the mixture to initiate a reaction. Afterreacting at 40 C for 30 minutes, 10 μl of 0.5 M EDTA was added theretoto terminate the reaction. Absorbance at 260 nm was then measured. As aresult, the absorbance at 260 nm from a reaction in which a cell crudeextract prepared from Escherichia coli HB101 harboring pRHB11 was usedwas clearly higher than that from a reaction in which a cell crudeextract prepared from Escherichia coli HB101 harboring pRHB1 was used.Thus, it was demonstrated that pRHB11 contained an RNase H gene and thatEscherichia coli harboring pRHB11 expressed an RNase H activity.

(6) Preparation of Purified RNase HII Preparation

Escherichia coli HB101 transformed with pRHB11 obtained in ReferentialExample 2-(4) was inoculated into 1 L of LB medium containing 100 μg/mlof ampicillin and cultured with shaking at 37 C for 16 hours. Aftercultivation, cells collected by centrifugation were suspended in 52.3 mlof a sonication buffer [50 mM tris-HCl (pH 8.0), 2 mM 2-mercaptoethanol,10% glycerol, 2 mM phenylmethanesulfonyl fluoride] and sonicated. Asupernatant obtained by centrifuging the sonicated suspension at 12000rpm for 10 minutes was heated at 60 C for 15 minutes. It was thencentrifuged at 12000 rpm for 10 minutes again to collect a supernatant.Thus, 50.0 ml of a heated supernatant was obtained.

The solution was subjected to RESOURSE Q column (Amersham PharmaciaBiotech) equilibrated with Buffer C [50 mM tris-HCl (pH 8.0), 2 mM2-mercaptoethanol, 10% glycerol] and chromatographed using FPLC system(Amersham Pharmacia Biotech). As a result, RNase HII flowed through theRESOURSE Q column. 51 ml of the flow-through RNase HII fraction wassubjected to RESOURSE S column (Amersham Pharmacia Biotech) equilibratedwith Buffer C and eluted with a linear gradient of 0 to 500 mM NaClusing FPLC system. A fraction containing RNase II eluted with about 240mM NaCl was obtained. 3.0 ml of the RNase II fraction was subjected intwo portions to PD-10 column (Amersham Pharmacia Biotech) equilibratedwith Buffer C containing 50 mM NaCl. 7.0 ml the resulting eluate wassubjected to HiTrap-heparin column (Amersham Pharmacia Biotech)equilibrated with Buffer C containing 50 mM NaCl and eluted with alinear gradient of 50 to 550 mM NaCl using FPLC system. A fractioncontaining RNase II eluted with about 310 mM NaCl was obtained. 4.4 mlof the RNase II fraction was concentrated by ultrafiltration usingCentricon-10 (Amicon). 280 μl of the concentrate was subjected toSuperdex 200 gel filtration column (Amersham Pharmacia Biotech)equilibrated with 50 mM tris-HCl (pH 8.0) containing 100 mM NaCl and 0.1mM EDTA and eluted with the same buffer. As a result, RNase HII waseluted at a position corresponding to a molecular weight of 35kilodalton. This molecular weight corresponds to that of RNase HII in aform of a monomer. The thus eluted RNase HII was used as Bca RNase HIIpreparation.

The enzymatic activity of the thus obtained Bca RNase HII preparationwas measured as follows.

100 μl of a reaction mixture [20 mM HEPES-potassium hydroxide (pH 7.8),0.01% bovine serum albumin (Takara Shuzo), 1% dimethyl sulfoxide, 10 mMmanganese chloride, 20 μg/ml poly(dT) (Amersham Pharmacia Biotech), 30μg/ml poly(rA) (Amersham Pharmacia Biotech)] which had been incubated at40 C was added to 1 μl of the Bca RNase HII preparation. The mixture wasreacted at 40 C for 10 minutes. The reaction was then terminated byadding 10 μl of 0.5 M EDTA (pH 8.0). Absorbance at 260 nm was thenmeasured.

As a result, an RNase H activity was observed for the Bca RNase HIIpreparation.

Referential Example 3 Cloning of Bacillus Caldotenax RNase HIII Gene

(1) Cloning of Fragment of RNase HIII Gene

Primers BsuIII-1, BsuIII-3, BsuIII-6 and BsuIII-8 represented by SEQ IDNOS:106-109 for screening a gene encoding RNase HIII were synthesizedbased on the amino acid sequences of regions well conserved amongBacillus subtilis and other organisms determined on the basis of thehomology among the amino acid sequences of RNase HIIIs from Bacillussubtilis [Biochemistry, 38:605-608 (1999)] and other organisms.

A first PCR was carried out in a volume of 50 μl using 200 ng of theBacillus caldotenax genomic DNA as prepared in Referential Example 2-(1)as a template, and 100 pmol of BsuIII-1 and 100 pmol of BsuIII-8 asprimers. A second PCR was then carried out in a volume of 100 μl using 1μl of the reaction mixture as a template, and 100 pmol of BsuIII-3 and100 pmol of BsuIII-6 as primers. TaKaRa Taq polymerase (Takara Shuzo)was used as a DNA polymerase for the two PCRs according to the attachedprotocol. The PCRs were carried out as follows: 25 (the first PCR) or 30(the second PCR) cycles of 94 C for 30 seconds, 45 C for 30 seconds and72 C for 1 minute.

An amplified DNA fragment of about 450 bp was blunt-ended using T4 DNApolymerase (Takara Shuzo) and then subjected to agarose gelelectrophoresis to recover the amplified DNA fragment of about 450 bp.The about 450-bp DNA fragment was ligated with pUC119 (Takara Shuzo)digested with Sma I (Takara Shuzo) using T4 DNA ligase (Takara Shuzo).The ligation mixture was used to transform Escherichia coli JM109. Theresulting transformants were cultured to obtain a plasmid pBCA3204 intowhich the about 450-bp DNA fragment was inserted.

(2) Cloning of RNase HIII Gene Using Southern Hybridization Method

The nucleotide sequence of the DNA fragment inserted in pBCA3204obtained in Referential Example 3-(1) was determined. Primers RNIII-S3and BcaRNIII-3 represented by SEQ ID NOS:110 and 111 were synthesized onthe basis of the determined nucleotide sequence. A PCR was carried outin a volume of 100 μl using RNIII-S3 and BcaRNIII-3 as primers andpBCA3204 as a template. TaKaRa Z-Taq (Takara Shuzo) was used as a DNApolymerase for the PCR according to the attached protocol. The PCR wascarried out as follows: 30 cycles of 98 C for 0 second, 55 C for 0second and 72 C for 20 seconds. After reaction, the reaction mixture wassubjected to phenol-chloroform extraction, ethanol precipitation andagarose gel electrophoresis to recover a DNA fragment of about 0.4 kbfrom the gel. The about 0.4-kb DNA fragment was labeled using DIG DNALabeling Kit (Boehringer Mannheim) to prepare a probe.

20 μg of the Bacillus caldotenax genomic DNA prepared in ReferentialExample 2-(1) was completely digested with BamHI, EcoRI, HindIII, PstIor XbaI (all from Takara Shuzo). The half of each of the digests wasthen subjected to agarose gel electrophoresis. The DNAs were transferredfrom the agarose gel to a nylon membrane using 0.4 N sodium hydroxideand fixed at 120 C for 30 minutes. The membrane was pre-incubated in asealed bag containing 30 ml of a hybridization buffer [43.4 g/L sodiumchloride, 17.6 g/L sodium citrate, 1% blocking agent (BoehringerMannheim), 0.1% N-lauroyl sarcosine, 0.02% sodium lauryl sulfate (SDS)]at 60 C for 4 hours and then incubated in a sealed bag containing 5 mlof a hybridization buffer containing the probe at 60 C for 16 hours.

The membrane was washed twice in 50 ml of 2×SSC (17.5 g/L NaCl, 8.8 g/Lsodium citrate) containing 0.1% SDS at room temperature, and twice in 50ml of 0.5×SSC (4.3 g/L sodium chloride, 1.9 g/L sodium citrate)containing 0.1% SDS at 45 C. Then, an EcoRI fragment of about 8 kb, aPstI fragment of about 4.5 kb and a HindIII fragment of about 1 kb whichhave sequences complementary to the probe were detected using DIGnucleic acid detection kit (Boehringer Mannheim).

The remaining half of the Bacillus caldotenax genomic DNA completelydigested with PstI was subjected to agarose gel electrophoresis. PstIfragments of about 4.5 kb were recovered from the gel. The DNA fragmentswere then ligated with a plasmid vector pTV119N, which had been digestedwith PstI and dephosphorylated with alkaline phosphatase (Takara Shuzo).The ligation mixture was used to transform Escherichia coli JM109.

A PCR was carried out in a volume of 50 μl using RNIII-S3 and BcaRNIII-3as primers, and one of the colonies as a template to select a colonypresumably harboring an RNase HIII gene. TaKaRa-Z Taq (Takara Shuzo) wasused for the PCR according to the attached protocol. The PCR was carriedout as follows: 30 cycles of 98 C for 0 second, 55 C for 0 second and 72C for 20 seconds. As a result, it was found that the gene of interestwas contained in the colony No. 88.

A PCR was carried out using a plasmid prepared from the colony No. 88 asa template, and a primer pair RN-N (Takara Shuzo) and BcaRNIII-3 or aprimer pair M4 (Takara Shuzo) and RNIII-S3 to examine whether or not theentire RNase HIII gene was contained in the plasmid. As a result, it wasfound that the entire RNase HIII gene was contained in the plasmid,which was designated as pBCA3P88.

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HIII Gene

The nucleotide sequence of the DNA fragment inserted into the plasmidpBCA3P88 obtained in Referential Example 3-(2) was determined accordingto a dideoxy method.

Analysis of the determined nucleotide sequence revealed the existence ofan open reading frame encoding an amino acid sequence including theN-terminal amino acid sequence of RNase HIII. The nucleotide sequence ofthe open reading frame and the amino acid sequence of RNase HIII deducedfrom the nucleotide sequence are shown in SEQ ID NO:112 and SEQ IDNO:113, respectively.

(4) Construction of Plasmid for Expressing RNase HIII

A PCR was carried out in a volume of 100 μl using the plasmid pBCA3P88as described in Referential Example 3-(2) as a template, BcaRNIIINderepresented by SEQ ID NO:114 designed with reference to the sequencearound the above-mentioned open reading frame for RNase HIII and M13primer M4 (Takara Shuzo). Pyrobest DNA polymerase (Takara Shuzo) wasused as a DNA polymerase for the PCR according to the attached protocol.The PCR was carried out as follows: 30 cycles of 94 C for 30 seconds, 55C for 30 seconds and 72 C for 3 minutes. An amplified DNA fragment ofabout 4 kb was digested with NdeI (Takara Shuzo) and subjected toagarose gel electrophoresis to recover an NdeI fragment of about 1.4 kbfrom the gel. The about 1.4-kb DNA fragment was ligated with pTV119Nd (aplasmid in which the NcoI site in pTV119N is converted into a NdeI site)which had been digested with NdeI and dephosphorylated with alkalinephosphatase (Takara Shuzo). The ligation mixture was used to transformEscherichia coli JM109.

Next, a PCR was carried out in a volume of 50 μl using one of thecolonies as a template, and RN-N (Takara Shuzo) and BcaRNIII-3 asprimers in order to screen for a plasmid in which the RNase HIII gene inthe NdeI fragment was linked downstream from the lac promoter in thevector pTV119Nd. A colony presumably harboring the RNase HIII gene wasthen selected. TaKaRa-Z Taq (Takara Shuzo) was used as a DNA polymerasefor the PCR according to the attached protocol. The PCR was carried outas follows: 30 cycles of 98 C for 0 second, 55 C for 0 second and 72 Cfor 20 seconds. As a result, it was found that the colony No. 2contained a plasmid in which the RNase HIII gene in the NdeI fragmentwas linked downstream from the lac promoter in the vector pTV119Nd. Thisplasmid was designated as pBCA3Nd2.

The determination of the nucleotide sequence of the DNA fragmentinserted into the plasmid by a dideoxy method revealed that there was nomutation due to the PCR except that the initiation codon GTG was changedto ATG.

Escherichia coli JM109 transformed with the plasmid pBCA3Nd2 isdesignated and indicated as Escherichia coli JM109/pBCA3Nd2, anddeposited on Sep. 5, 2000 at the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology under accession number FERM P-18019.

(5) Preparation of Purified RNase HIII Preparation

Escherichia coli JM109 transformed with pBCA3Nd2 obtained in ReferentialExample 3-(4) was inoculated into 2 L of LB medium containing 100 μg/mlof ampicillin and cultured with shaking at 37 C for 16 hours. Aftercultivation, cells collected by centrifugation were suspended in 39.6 mlof a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12000 rpm for 10 minutes washeated at 60 C for 15 minutes. It was then centrifuged at 12000 rpm for10 minutes again to collect a supernatant. Thus, 39.8 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HIII flowed through the RESOURSE Q column.

45 ml of the flow-through RNase HIII fraction was dialyzed against 2 Lof Buffer B [50 mM tris-HCl (pH 7.0), 1 mM EDTA] for 2 hours. Thedialysis was repeated for two more times under the same conditions. 55.8ml of the dialyzed enzyme solution was subjected to RESOURSE S column(Amersham Pharmacia Biotech) equilibrated with Buffer B and eluted witha linear gradient of 0 to 500 mM NaCl using FPLC system. A fractioncontaining RNase HIII eluted with about 105 mM NaCl was obtained.

Buffer B containing 1 M NaCl was added to 7.0 ml of the fraction to makethe NaCl concentration to 150 mM. The mixture was subjected toHiTrap-heparin column (Amersham Pharmacia Biotech) equilibrated withBuffer B containing 150 mM NaCl. As a result, RNase HIII flowed throughthe HiTrap-heparin column.

7.5 ml of the flow-through RNase HIII fraction was concentrated byultrafiltration using Centricon-10 (Amicon). 190 μl of the concentratewas subjected to Superdex 200 gel filtration column (Amersham PharmaciaBiotech) equilibrated with 50 mM tris-HCl (pH 7.0) containing 100 mMNaCl and 0.1 mM EDTA and eluted with the same buffer. As a result, RNaseHIII was eluted at a position corresponding to a molecular weight of 33kilodalton. This molecular weight corresponds to that of RNase HIII in aform of a monomer.

The thus eluted RNase HIII was used as Bca RNase HIII preparation.

The enzymatic activity of the thus obtained Bca RNase HIII preparationwas measured as follows.

100 μl of a reaction mixture [20 mM HEPES-potassium hydroxide (pH 7.8),0.01% bovine serum albumin (Takara Shuzo), 1% dimethyl sulfoxide, 4 mMmagnesium acetate, 20 μg/ml poly(dT) (Amersham Pharmacia Biotech), 30μg/ml poly(rA) (Amersham Pharmacia Biotech)] which had been incubated at40 C was added to 1 μl of the Bca RNase HIII preparation. The mixturewas reacted at 40 C for 10 minutes. The reaction was terminated byadding 10 μl of 0.5 M EDTA (pH 8.0). Absorbance at 260 nm was thenmeasured. As a result, an RNase H activity was observed for the BcaRNase HIII preparation.

Referential Example 4 Cloning of Pyrococcus furiosus RNase HII Gene

(1) Preparation of Genomic DNA from Pyrococcus furiosus

2 L of a medium containing 1% Tryptone (Difco Laboratories), 0.5% yeastextract (Difco Laboratories), 1% soluble starch (Nacalai Tesque), 3.5%Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid (JamarineLaboratory), 0.003% MgSO₄, 0.001% NaCl, 0.0001% FeSO₄.7H₂O, 0.0001%COSO₄, 0.0001% CaCl₂.7H₂O, 0.0001% ZnSO₄, 0.1 ppm CuSO₄.5H₂O, 0.1 ppmKAl(SO₄)₂, 0.1 ppm H₃BO₄, 0.1 ppm Na₂MoO₄.2H₂O and 0.25 ppm NiCl₂.6H₂Owas placed in a 2 L medium bottle, sterilized at 120 C for 20 minutes,bubbled with nitrogen gas to remove dissolved oxygen, then Pyrococcusfuriosus (purchased from Deutsche Sammlung von Mikroorganismen; DSM3638)was inoculated into the medium and cultured at 95 C for 16 hours withoutshaking. After cultivation, cells were collected by centrifugation.

The resulting cells were then suspended in 4 ml of 25% sucrose, 50 mMtris-HCl (pH 8.0). 0.4 ml of 10 mg/ml lysozyme chloride (Nacalai Tesque)in water was added thereto. The mixture was reacted at 20 C for 1 hour.After reaction, 24 ml of a mixture containing 150 mM NaCl, 1 mM EDTA and20 mM tris-HCl (pH 8.0), 0.2 ml of 20 mg/ml proteinase K (Takara Shuzo)and 2 ml of 10% aqueous solution of sodium lauryl sulfate and were addedto the reaction mixture. The mixture was incubated at 37 C for 1 hour.After reaction, the mixture was subjected to phenol-chloroformextraction followed by ethanol precipitation to prepare about 1 mg ofgenomic DNA.

(2) Cloning of RNase HII Gene

The entire genomic sequence of Pyrococcus horikoshii was published [DNAResearch, 5:55-76 (1998)]. The existence of a gene encoding a homologueof RNase HII (PH1650) in the genome was known (SEQ ID NO:115, the homepage of National Institute of Technology and Evaluation, Ministry ofInternational Trade and Industry of Japan: http://www/nite.go.jp/).

Homology between the PH1650 gene and the partially published genomicsequence of Pyrococcus furiosus (the home page of University of Utah,Utah Genome Center: http://www.genome.utah.edu/sequence.html) wassearched. As a result, a highly homologous sequence was found. Primers1650Nde (SEQ ID NO:116) and 1650Bam (SEQ ID NO:117) were synthesized onthe basis of the homologous sequence.

A PCR was carried out in a volume of 100 μl using 200 ng of thePyrococcus furiosus genomic DNA obtained in Referential Example 4-(1) asa template, and 20 pmol of 1650Nde and 20 pmol of 1650Bam as primers.TaKaRa Ex Taq (Takara Shuzo) was used as a DNA polymerase for the PCRaccording to the attached protocol. The PCR was carried out as follows:30 cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 C for 1minute. An amplified DNA fragment of about 0.7 kb was digested with NdeIand BamHI (both from Takara Shuzo). The resulting DNA fragment wasinserted between the NdeI site and the BamHI site in a plasmid vectorpET3a (Novagen) to make a plasmid pPFU220.

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequence of the DNA fragment inserted into pPFU220obtained in Referential Example 4-(2) was determined according to adideoxy method.

Analysis of the determined nucleotide sequence revealed the existence ofan open reading frame presumably encoding RNase HII. The nucleotidesequence of the open reading frame is shown in SEQ ID NO:118. The aminoacid sequence of RNase HII deduced from the nucleotide sequence is shownin SEQ ID NO:119.

Escherichia coli JM109 transformed with the plasmid pPFU220 isdesignated and indicated as Escherichia coli JM109/pPFU220, anddeposited on Sep. 5, 2000 at the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology under accession number FERM P-18020.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli HMS174(DE3) (Novagen) was transformed with pPFU220obtained in Referential Example 4-(2). The resulting Escherichia coliHMS174(DE3) harboring pPFU220 was inoculated into 2 L of LB mediumcontaining 100 μg/ml of ampicillin and cultured with shaking at 37 C for16 hours. After cultivation, cells collected by centrifugation weresuspended in 66.0 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 1mM EDTA, 2 mM phenylmethanesulfonyl fluoride] and sonicated. Asupernatant obtained by centrifuging the sonicated suspension at 12000rpm for 10 minutes was heated at 60 C for 15 minutes. It was thencentrifuged at 12000 rpm for 10 minutes again to collect a supernatant.Thus, 61.5 ml of a heated supernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

60.0 ml of the flow-through RNase HII fraction was subjected to RESOURSES column (Amersham Pharmacia Biotech) equilibrated with Buffer A andeluted with a linear gradient of 0 to 500 mM NaCl using FPLC system. Afraction containing RNase HII eluted with about 150 mM NaCl wasobtained. 2.0 ml of the RNase HII fraction was concentrated byultrafiltration using Centricon-10 (Amicon). 250 μl of the concentratewas subjected to Superdex 200 gel filtration column (Amersham PharmaciaBiotech) equilibrated with 50 mM tris-HCl (pH 8.0) containing 100 mMNaCl and 0.1 mM EDTA and eluted with the same buffer. As a result, RNaseHII was eluted at a position corresponding to a molecular weight of 17kilodalton. This molecular weight corresponds to that of RNase HII in aform of a monomer.

The thus eluted RNase HII was used as Pfu RNase HII preparation.

The enzymatic activity of the thus obtained Pfu RNase HII preparationwas measured as described in Referential Example 3-(5). As a result, anRNase H activity was observed for the Pfu RNase HII preparation.

Referential Example 5 Cloning of Thermotoga maritima RNase HII Gene

(1) Preparation of Genomic DNA from Thermotoga maritima

2 L of a medium containing 1% Tryptone, 0.5% yeast extract, 1% solublestarch, 3.5% Jamarine S Solid, 0.5% Jamarine S Liquid, 0.003% MgSO₄,0.001% NaCl, 0.0001% FeSO₄.7H₂O, 0.0001% COSO₄, 0.0001% CaCl₂.7H₂O,0.0001% ZnSO₄, 0.1 ppm CuSO₄.5H₂O, 0.1 ppm KAl(SO₄)₂, 0.1 ppm H₃BO₃, 0.1ppm Na₂MoO₄.2H₂O and 0.25 ppm NiCl₂.6H₂O was placed in a 2 L mediumbottle, sterilized at 120 C for 20 minutes, bubbled with nitrogen gas toremove dissolved oxygen, then Thermotoga maritima (purchased fromDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSM3109)was inoculated into the medium and cultured at 85 C for 16 hours withoutshaking.

Cells collected by centrifugation from 300 ml of the culture were thensuspended in 3 ml of TE buffer [10 mM tris-HCl (pH 7.5), 1 mM EDTA]. 150μl of 10% aqueous solution of sodium lauryl sulfate (Nacalai Tesque) and15 μl of 20 mg/ml proteinase K (Takara Shuzo) were added thereto. Themixture was incubated at 37 C for 1 hour. After reaction, 0.5 ml of 5 MNaCl was added to the mixture. After thoroughly mixing, 0.4 ml of aCTAB-NaCl solution [10% cetyltrimethylammonium bromide (Nacalai Tesque),0.7 M NaCl] was added to the mixture. After thoroughly mixing, themixture was incubated at 65 C for 10 minutes. 1.5 ml of a mixture ofchloroform/isoamyl alcohol (24:1, v/v) was added thereto. The mixturewas gently mixed for 10 minutes and centrifuged at 20000×g for 5minutes. After centrifugation, an equal volume of a mixture of phenolsaturated with 100 mM tris-HCl (pH 8.0)/chloroform/isoamyl alcohol(25:24:1, v/v) was added to the resulting supernatant. The mixture wasgently mixed for 10 minutes and then centrifuged at 20000×g for 5minutes. After centrifugation, 0.6 volume of 2-propanol was added to thesupernatant. The precipitate obtained by centrifugation at 10000×g for 5minutes was washed with 70% ethanol in water, air-dried and thendissolved in 200 μl of TE to obtain a genomic DNA solution.

(2) Cloning of RNase HII Gene

Oligonucleotides 915-F1, 915-F2, 915-R1 and 915-R2 represented by SEQ IDNOS:120-123 were synthesized on the basis of the nucleotide sequence ofa portion that had been identified as an RNase HII gene in thenucleotide sequence of the genomic DNA of Thermotoga maritima(http://www.tigr.org/tdb/CMR/btm/htmls/SplashPage.html) in order toobtain an amplified DNA fragment containing an RNase H gene by carryingout a PCR using the Thermotoga maritima genomic DNA as a template.

PCRs were carried out using the Thermotoga maritima genomic DNA asprepared in Referential Example 5-(1) as a template, and 915-F1 and915-R1, 915-F1 and 915-R2, 915-F2 and 915-R1, or 915-F2 and 915-R2 as aprimer pair. TaKaRa Ex Taq was used as a DNA polymerase for the PCRsaccording to the attached protocol. The PCRs were carried out asfollows: 25 cycles of 95 C for 0.5 minute, 55 C for 0.5 minute and 72 Cfor 1.5 minute. After reactions, the respective PCR products weresubjected to agarose gel electrophoresis to extract and purify amplifiedDNA fragments of about 0.7 kb. The DNAs amplified using a primer pair915-F1 and 915-R1 or 915-F1 and 915-R2 were digested with HindIII andXbaI (both from Takara Shuzo) and ligated with pUC19 digested withHindIII and XbaI using T4 DNA ligase. The ligation mixture was used totransform Escherichia coli JM109. The resulting transformants werecultured to prepare plasmid DNAs into which the about 0.7-kb DNAs wereinserted. As a result, plasmids No. 1 and No. 2 having DNAs amplifiedusing 915-F1 and 915-R1, and plasmids No. 3 and No. 4 having DNAsamplified using 915-F1 and 915-R2 were obtained.

In addition, the DNAs amplified using a primer pair 915-F2 and 915-R1 or915-F2 and 915-R2 were doubly digested with NcoI (Takara Shuzo) and XbaIand ligated with pTV119N (Takara Shuzo) doubly digested with NcoI andXbaI using T4 DNA ligase. The ligation mixture was used to transformEscherichia coli JM109.

The resulting transformants were cultured to prepare plasmid DNAs intowhich the about 0.7-kb DNAs were inserted. As a result, plasmids No. 5and No. 6 having DNAs amplified using 915-F2 and 915-R1, and a plasmidNo. 7 having a DNA amplified using 915-F2 and 915-R2 were obtained.

Escherichia coli JM109 transformed with the plasmid No. 7 is designatedand indicated as Escherichia coli JM109/pTM-RNH, and deposited on Sep.5, 2000 at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology under accessionnumber FERM P-18018.

(3) Expression of Thermotoga maritima RNase HII Gene

Escherichia coli JM109 transformed with one of the plasmids No. 1 to 7or pUC19 was inoculated into 5 ml of LB medium (10 g/L Tryptone, 5 g/Lyeast extract, 5 g/L NaCl, pH 7.2) containing 100 μg/ml of ampicillinand cultured with shaking at 37 C. When the absorbance at 660 nm reached0.5, isopropyl-β-D-thiogalactopyranoside was added thereto to a finalconcentration of 1 mM and the cells were cultured overnight. Aftercultivation, cells collected by centrifugation were suspended in 1 ml ofTE buffer and sonicated. The sonicated suspension was heated at 80 C for10 minute. A supernatant obtained by centrifugation was used as a cellcrude extract. Absorbance was measured using the cell crude extract asdescribed in Referential Example 2-(5). As a result, when reactions werecarried out in the presence of MnCl₂, the absorbance at 260 nm from eachof the reactions in which cell crude extracts prepared from Escherichiacoli JM109 harboring the plasmid No. 3, 5, 6 or 7 were used was clearlyhigher than that from a reaction in which a crude extract prepared fromEscherichia coli JM109 harboring pUC19 was used. Thus, it wasdemonstrated that the plasmids No. 3, 5, 6 and 7 contained RNase H genesand that Escherichia coli harboring one of these plasmids expressed anRNase H activity.

The partial nucleotide sequences of the DNA fragments inserted into theplasmids which were demonstrated to express RNase activities inEscherichia coli as described above were determined. Analysis of thedetermined nucleotide sequences revealed an open reading framepresumably encoding RNase HII. The nucleotide sequence of the openreading frame is shown in SEQ ID NO:232. The amino acid sequence ofRNase HII deduced from the nucleotide sequence is shown in SEQ IDNO:233. Then, it was found that one base substitution that waspresumably generated upon the PCR was observed in a portion of thenucleotide sequence of the DNA fragment inserted in the plasmid No. 7,resulting in the change in the encoded amino acid residue.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli JM109 was transformed with pTM-RNH obtained inReferential Example 5-(2). The resulting Escherichia coli JM109harboring pTM-RNH was inoculated into 1 L of LB medium containing 100μg/ml of ampicillin and cultured with shaking at 37 C for 16 hours.After cultivation, cells collected by centrifugation were suspended in31.0 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 2 mM2-mercaptoethanol, 10% glycerol, 2 mM phenylmethanesulfonyl fluoride]and sonicated. A supernatant obtained by centrifuging the sonicatedsuspension at 12000 rpm for 10 minutes was heated at 70 C for 15minutes. It was then centrifuged at 12000 rpm for 10 minutes again tocollect a supernatant. Thus, 32.0 ml of a heated supernatant wasobtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer C [50 mM tris-HCl (pH 8.0),2 mM 2-mercaptoethanol, 10% glycerol] and chromatographed using FPLCsystem (Amersham Pharmacia Biotech). As a result, RNase HII flowedthrough the RESOURSE Q column. 32.5 ml of the flow-through RNase HIIfraction was subjected to RESOURSE S column (Amersham Pharmacia Biotech)equilibrated with Buffer C and eluted with a linear gradient of 0 to 500mM NaCl using FPLC system. A fraction containing RNase II eluted withabout 240 mM NaCl was obtained. 2.0 ml of the RNase II fraction wassubjected to PD-10 column (Amersham Pharmacia Biotech) equilibrated withBuffer C containing 50 mM NaCl. 3.5 ml of the resulting eluate wassubjected to HiTrap-heparin column (Amersham Pharmacia Biotech)equilibrated with Buffer C containing 50 mM NaCl and eluted with alinear gradient of 50 to 550 mM NaCl using FPLC system. As a result, afraction containing RNase II eluted with about 295 mM NaCl was obtained.The thus eluted RNase HII was used as Tma RNase HII preparation.

The enzymatic activity of the thus obtained Tma RNase HII preparationwas measured as described in Referential Example 2-(6). As a result, anRNase H activity was observed for the Tma RNase HII preparation.

Referential Example 6 Cloning of RNase HII Gene from Pyrococcushorikoshii

(1) Preparation of Genomic DNA from Pyrococcus horikoshii

2 L of a medium containing 1% Tryptone (Difco Laboratories), 0.5% yeastextract (Difco Laboratories), 1% soluble starch (Nacalai Tesque), 3.5%Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid (JamarineLaboratory), 0.003% MgSO₄, 0.001% NaCl, 0.0001% FeSO₄.7H₂O, 0.0001%CoSO₄, 0.0001% CaCl₂.7H₂O, 0.0001% ZnSO₄, 0.1 ppm CuSO₄.5H₂O, 0.1 ppmKAl(SO₄)₂, 0.1 ppm H₃BO₄, 0.1 ppm Na₂MoO₄.2H₂O and 0.25 ppm NiCl₂.6H₂Owas placed in a 2-L medium bottle, sterilized at 120 C for 20 minutes,bubbled with nitrogen gas to remove dissolved oxygen, then Pyrococcushorikoshii OT3 (purchased from the Institute of Physical and ChemicalResearch (RIKEN); JCM9974) was inoculated into the medium and culturedat 95 C for 16 hours without shaking. After cultivation, cells werecollected by centrifugation.

The cells were then suspended in 4 ml of 25% sucrose, 50 mM tris-HCl (pH8.0). 0.4 ml of 10 mg/ml lysozyme chloride (Nacalai Tesque) in water wasadded thereto. The mixture was reacted at 20 C for 1 hour. Afterreaction, 24 ml of a mixture containing 150 mM NaCl, 1 mM EDTA and 20 mMtris-HCl (pH 8.0), 0.2 ml of 20 mg/ml proteinase K (Takara Shuzo) and 2ml of 10% aqueous solution of sodium lauryl sulfate were added to thereaction mixture. The mixture was incubated at 37 C for 1 hour.

After reaction, the mixture was subjected to phenol-chloroformextraction followed by ethanol precipitation to prepare about 1 mg ofgenomic DNA.

(2) Cloning of RNase HII Gene

The entire genomic sequence of the Pyrococcus horikoshii has beenpublished [DNA Research, 5:55-76 (1998)]. The existence of one geneencoding a homologue of RNase HII (PH1650) was known (SEQ ID NO:234, thehome page of National Institute of Technology and Evaluation:http://www/nite.go.jp/).

Primers PhoNde (SEQ ID NO:235) and PhoBam (SEQ ID NO:236) weresynthesized on the basis of the sequence of the PH1650 gene (SEQ IDNO:234).

A PCR was carried out using 100 ng of the Pyrococcus horikoshii genomicDNA prepared in Referential Example 6-(1) as a template, and 20 pmoleach of PhoNde and PhoBam as primers in a volume of 100 μl. TaKaRa ExTaq (Takara Shuzo) was used as a DNA polymerase for the PCR according tothe attached protocol. The PCR was carried out as follows: 40 cycles of94 C for 30 seconds, 55 C for 30 seconds and 72 C for 1 minute. Anamplified DNA fragment of about 0.7 kb was digested with NdeI and BamHI(both from Takara Shuzo). Then, a plasmid pPHO238 was constructed byincorporating the resulting DNA fragment between NdeI and BamHI sites ina plasmid vector pET3a (Novagen).

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequence of the DNA fragment inserted into pPHO238obtained in Referential Example 6-(2) was determined according to adideoxy method.

Analysis of the determined nucleotide sequence revealed an open readingframe presumably encoding RNase HII. The nucleotide sequence of the openreading frame is shown in SEQ ID NO:237. The amino acid sequence ofRNase HII deduced from the nucleotide sequence is shown in SEQ IDNO:238.

Escherichia coli JM109 transformed with the plasmid pPHO238 isdesignated and indicated as Escherichia coli JM109/pPHO238, anddeposited on Feb. 22, 2001 at International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology underaccession number FERM P-18222.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli HMS174(DE3) (Novagen) was transformed with pPHO238obtained in Referential Example 6-(2). The resulting Escherichia coliHMS174(DE3) harboring pPHO238 was inoculated into 1 L of LB mediumcontaining 100 μg/ml of ampicillin and cultured with shaking at 37 C for16 hours. After cultivation, cells collected by centrifugation weresuspended in 34.3 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 1mM EDTA, 2 mM phenylmethanesulfonyl fluoride] and sonicated. Asupernatant obtained by centrifuging the sonicated suspension at 12000rpm for 10 minutes was heated at 80 C for 15 minutes. It was thencentrifuged at 12000 rpm for 10 minutes again to collect a supernatant.Thus, 33.5 ml of a heated supernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

35.0 ml of the flow-through RNase HII fraction was dialyzed against 2 Lof Buffer B (50 mM tris-HCl (pH 7.0), 1 mM EDTA) for 2 hours. Thedialysis was repeated two more times. 34.5 ml of the dialyzed enzymesolution was subjected to RESOURSE S column (Amersham Pharmacia Biotech)equilibrated with Buffer B and eluted with a linear gradient of 0 to 500mM NaCl using FPLC system. A fraction containing RNase HII eluted withabout 155 mM NaCl was obtained.

Buffer B was added to 4.0 ml of the fraction to make the NaClconcentration to 50 mM. The mixture was subjected to HiTrap-heparincolumn (Amersham Pharmacia Biotech) equilibrated with Buffer Bcontaining 50 mM NaCl and eluted with a linear gradient of 50 to 550 mMNaCl using FPLC system. As a result, a fraction containing RNase HIIeluted with about 160 mM NaCl was obtained.

6.9 ml of the RNase HII fraction was concentrated by ultrafiltrationusing Centricon-10 (Amicon). Two portions each separated from 250 μl ofthe concentrate were subjected to Superose 6 gel filtration column(Amersham Pharmacia Biotech) equilibrated with 50 mM tris-HCl (pH 7.0)containing 100 mM NaCl and 0.1 mM EDTA and eluted with the same buffer.As a result, RNase HII was eluted at a position corresponding to amolecular weight of 24.5 kilodalton. This molecular weight correspondsto that of RNase HII in a form of a monomer.

The RNase HII eluted as described above was used as Pho RNase HIIpreparation.

The enzymatic activity of the thus obtained Pho RNase HII preparationwas measured as described in Referential Example 3-(5). As a result, anRNase H activity was observed for the Pho RNase HII preparation.

Referential Example 7 Cloning of RNase HII Gene from Archaeoglobusfulgidus

(1) Preparation of Genomic DNA from Archaeoglobus fulgidus

Cells of Archaeoglobus fulgidus (purchased from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH; DSM4139) collected from 8 ml of aculture was suspended in 100 μl of 25% sucrose, 50 mM tris-HCl (pH 8.0).20 μl of 0.5 M EDTA and 10 μl of 10 mg/ml lysozyme chloride (NacalaiTesque) in water was added thereto. The mixture was reacted at 20 C for1 hour. After reaction, 800 μl of a mixture containing 150 mM NaCl, 1 mMEDTA and 20 mM tris-HCl (pH 8.0), 10 μl of 20 mg/ml proteinase K (TakaraShuzo) and 50 μl of 10% aqueous solution of sodium lauryl sulfate andwere added to the reaction mixture. The mixture was incubated at 37 Cfor 1 hour. After reaction, the mixture was subjected tophenol-chloroform extraction, ethanol precipitation and air-drying, andthen dissolved in 50 μl of TE to obtain a genomic DNA solution.

(2) Cloning of RNase HII Gene

The entire genomic sequence of the Archaeoglobus fulgidus has beenpublished [Nature, 390:364-370 (1997)]. The existence of one geneencoding a homologue of RNase HII (AF0621) was known (SEQ ID NO:239,http://www.tigr.org/tdb/CMR/btm/htmls/SplashPage.htlm).

Primers AfuNde (SEQ ID NO:240) and AfuBam (SEQ ID NO:241) weresynthesized on the basis of the sequence of the AF0621 gene (SEQ IDNO:239).

A PCR was carried out using 30 ng of the Archaeoglobus fulgidus genomicDNA prepared in Referential Example 7-(1) as a template, and 20 pmoleach of AfuNde and AfuBam as primers in a volume of 100 μl. Pyrobest DNApolymerase (Takara Shuzo) was used as a DNA polymerase for the PCRaccording to the attached protocol. The PCR was carried out as follows:40 cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 C for 1minute. An amplified DNA fragment of about 0.6 kb was digested with NdeIand BamHI (both from Takara Shuzo). Then, a plasmid pAFU204 wasconstructed by incorporating the resulting DNA fragment between NdeI andBamHI sites in a plasmid vector pTV119Nd (a plasmid in which the NcoIsite in pTV119N is converted into a NdeI site).

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequence of the DNA fragment inserted into pAFU204obtained in Referential Example 7-(2) was determined according to adideoxy method.

Analysis of the determined nucleotide sequence revealed an open readingframe presumably encoding RNase HII. The nucleotide sequence of the openreading frame is shown in SEQ ID NO:242. The amino acid sequence ofRNase HII deduced from the nucleotide sequence is shown in SEQ IDNO:243.

Escherichia coli JM109 transformed with the plasmid pAFU204 isdesignated and indicated as Escherichia coli JM109/pAFU204, anddeposited on Feb. 22, 2001 at International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology underaccession number FERM P-18221.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli JM109 was transformed with pAFU204 obtained inReferential Example 7-(2). The resulting Escherichia coli JM109harboring pAFU204 was inoculated into 2 L of LB medium containing 100μg/ml of ampicillin and cultured with shaking at 37 C for 16 hours.After cultivation, cells collected by centrifugation were suspended in37.1 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12000 rpm for 10 minutes washeated at 70 C for 15 minutes. It was then centrifuged at 12000 rpm for10 minutes again to collect a supernatant. Thus, 40.3 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

The flow-through RNase HII fraction was subjected to RESOURSE S column(Amersham Pharmacia Biotech) equilibrated with Buffer A andchromatographed using FPLC system (Amersham Pharmacia Biotech). As aresult, RNase HII flowed through the RESOURSE S column.

40.0 ml of the flow-through RNase HII fraction was dialyzed against 2 Lof Buffer B (50 mM tris-HCl (pH 7.0), 1 mM EDTA) containing 50 mM NaClfor 2 hours. The dialysis was repeated two more times. 40.2 ml of thedialyzed enzyme solution was subjected to HiTrap-heparin column(Amersham Pharmacia Biotech) equilibrated with Buffer B containing 50 mMNaCl and eluted with a linear gradient of 50 to 550 mM NaCl using FPLCsystem. As a result, a fraction containing RNase HII eluted with about240 mM NaCl was obtained.

7.8 ml of the RNase HII fraction was concentrated by ultrafiltrationusing Centricon-10 (Amicon). Four portions each separated from about 600μl of the concentrate were subjected to Superose 6 gel filtration column(Amersham Pharmacia Biotech) equilibrated with 50 mM tris-HCl (pH 7.0)containing 100 mM NaCl and 0.1 mM EDTA and eluted with the same buffer.As a result, RNase HII was eluted at a position corresponding to amolecular weight of 30.0 kilodalton. This molecular weight correspondsto that of RNase HII in a form of a monomer.

The RNase HII eluted as described above was used as Afu RNase HIIpreparation.

The enzymatic activity of the thus obtained Afu RNase HII preparationwas measured as described in Referential Example 3-(5). As a result, anRNase H activity was observed for the Afu RNase HII preparation.

Referential Example 8

Unit value of RNase H from Escherichi coli used in the method of thepresent invention was measured according to the following method.

(1) Preparation of Reagent Solutions Used

Reaction mixture for determining activity: The following substances atthe indicated final concentrations were contained in sterile water: 40mM tris-hydrochloride (pH 7.7 at 37 C), 4 mM magnesium chloride, 1 mMDTT, 0.003% BSA, 4% glycerol and 24 μM poly(dT).

Poly[8-³H]adenylic acid solution: 370 kBq of a poly[8-³H]adenylic acidsolution was dissolved in 200 μl of sterile water.

Polyadenylic acid solution: Polyadenylic acid was diluted to aconcentration of 3 mM with sterile ultrapure water.

Enzyme dilution solution: The following substances at the indicatedfinal concentrations were contained in sterile water: 25 mMtris-hydrochloride (pH 7.5 at 37 C), 5 mM 2-mercaptoethanol, 0.5 mM EDTA(pH 7.5 at 37 C), 30 mM sodium chloride and 50% glycerol.

Preparation of heat-denatured calf thymus DNA: 200 mg of calf thymus DNAwas suspended and allowed to swell in 100 ml of TE buffer. The solutionwas diluted to a concentration of 1 mg/ml with sterile ultrapure waterbased on the absorbance measured at UV 260 nm. The diluted solution washeated at 100° C. for 10 minutes and then rapidly cooled in an ice bath.

(2) Method for Measuring Activity

7 μl of the poly[8-³H]adenylic acid solution was added to 985 μl of thereaction mixture for determining activity prepared in (1) above. Themixture was incubated at 37° C. for 10 minutes. 8 μl of polyadenylicacid was added to the mixture to make the final concentration to 24 μM.The mixture was further incubated at 37° C. for 5 minutes. Thus, 1000 μlof a poly[8-³H]rA-poly-dT reaction mixture was prepared. 200 μl of thereaction mixture was then incubated at 30° C. for 5 minutes. 1 μl of anappropriate serial dilution of an enzyme solution was added thereto. 50μl each of samples was taken from the reaction mixture over time for usein subsequent measurement. The period of time in minutes from theaddition of the enzyme to the sampling is defined as Y. 50 μl of areaction mixture for total CPM or for blank was prepared by adding 1 μlof the enzyme dilution solution instead of an enzyme solution. 100 μl of100 mM sodium pyrophosphate, 50 μl of the heat-denatured calf thymus DNAsolution and 300 μl of 10% trichloroacetic acid (300 μl of ultrapurewater for measuring total CPM) were added to the sample. The mixture wasincubated at 0° C. for 5 minutes, and then centrifuged at 10000 rpm for10 minutes. After centrifugation, 250 μl of the resulting supernatantwas placed in a vial. 10 ml of Aquasol-2 (NEN Life Science Products) wasadded thereto. CPM was measured in a liquid scintillation counter.

(3) Calculation of Units

Unit value for each enzyme was calculated according to the followingequation.Unit/ml={(measuredCPM−blankCPM)×1.2*×20×1000×dilutionrate}200(μl)/(totalCPM×Y(min.)×50(μl)×9**)

-   -   1.2*: Amount in nmol of poly[8-³H]rA-poly-dT contained in total        CPM per 50 μl.    -   9**: Correction coefficient.

Example 1

(1) Synthesis of Template DNA and Primers

A single-stranded DNA of 99 bases as a template and primers used in thisExample were synthesized using a DNA synthesizer (Applied Biosystems).The nucleotide sequence of the single-stranded DNA of 99 bases is shownin

SEQ ID NO:1 of the Sequence Listing. The structures of the primers usedin this Example are described below in detail:

-   -   Primer Pair 1: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:2 or 3 of the Sequence Listing        and wholly composed of deoxyribonucleotides;    -   Primer Pair 2: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:4 or 5 of the Sequence Listing in        which the first and second nucleotides from the 3′-terminus are        ribonucleotides and the phosphate bond on the 5′-terminal side        of the second ribonucleotide from the 3′-terminus is replaced by        a phosphorothioate bond;    -   Primer Pair 3: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:6 or 7 of the Sequence Listing in        which the nucleotide at the 3′-terminus is a ribonucleotide and        the phosphate bond on the 5′-terminal side of the ribonucleotide        is replaced by a phosphorothioate bond;    -   Primer Pair 4: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:8 or 9 of the Sequence Listing in        which the first and second nucleotides from the 3′-terminus are        ribonucleotides; and    -   Primer Pair 5: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:10 or 11 of the Sequence Listing        in which the third and fourth nucleotides from the 3′-terminus        are ribonucleotides and the phosphate bond on the 5′-terminal        side of the fourth ribonucleotide form the 3′-terminus is        replaced by a phosphorothioate bond.

(2) Amplification Reaction

Bca BEST DNA polymerase (Takara Shuzo), which is a DNA polymeraselacking a 5′3′ exonuclease activity from Bacillus caldotenax, and clonedribonuclease H (Takara Shuzo), which is RNase H from E. coli, were usedto examine the reaction systems of Models 1 to 7 as described below.

A reaction mixture was prepared as follows.

35 mM tris-hydrochloride buffer (pH 7.5), 0.1 mg/ml bovine serum albumin(BSA), 2.7% glycerol, 5% dimethyl sulfoxide, 1.4 mM each of dNTPs, 10 mMmagnesium chloride, 20 pmol of one or both of the primers of one of theprimer pairs as described above in (1), 0.6 ng of the syntheticsingle-stranded DNA as the template, 5 U of Bca BEST DNA polymerase and60 U of cloned ribonuclease H to a final reaction volume of 50 μl. Thereaction mixture was mixed to homogeneity, incubated at 55° C. for 60minutes, and then heated at 90° C. for 2 minutes to inactivate theenzymes. 8 μl of the reaction mixture was subjected to electrophoresison 3% NuSieve 3:1 agarose (Takara Shuzo) gel. The primers used in therespective Models are described below:

-   -   Models 1-5: One of the Primer Pairs 1-5 was used;    -   Model 6: Only the downstream primer of the Primer Pair 2 was        used; and    -   Model 7: The Primer Pair 4 was used without the addition of        RNase H.

As a result, amplified fragments having a size of interest ranging fromabout 40 base pairs (bp) to about 90 bp were observed when the reactionmixtures of the Models 2 to 5 were used, indicating that DNAs areamplified using these reaction systems. An amplified fragment having anexpected size of about 70 bases (b) (a single-stranded DNA fragment) wasobserved for the Model 6 in which only one of the two primers was used.No DNA amplification was observed for the reaction of the Model 1 or 7.

(3) Confirmation of Amplification Products

The reaction mixture obtained by the reaction of the Model 4 asdescribed in (2) was filtrated using Microcon-100 (Takara Shuzo) torecover an amplified DNA fragment entrapped on the filter. Thenucleotide sequence of the DNA fragment was determined by the dideoxymethod. As a result, the fragment amplified by the above-mentionedreaction was confirmed to be a DNA having the same nucleotide sequenceas that of the DNA as the template.

(4) Examination of Reaction Time

The reaction mixture of the Model 2 as described above in (2) wasprepared to study the change in the amount of the amplification productwhen it was reacted for a varying time. The reaction mixture wasincubated for 0, 15, 30, 60, 90 or 120 minutes at 55° C. The mixture wasthen treated at 90° C. for 2 minutes to inactivate the enzymes. 8 μl ofthe reaction mixture was analyzed by electrophoresis on 3% NuSieve 3:1agarose gel. The results of the electrophoresis are shown in FIG. 41.Numbers 1 to 6 in the figure represent lanes to which the reactionmixture reacted for 0, 15, 30, 60, 90 or 120 minutes was applied,respectively. M represents a lane to which 100 bp DNA ladder marker(Takara Shuzo) was applied as a molecular weight marker.

As shown in FIG. 41, no amplification product was observed for areaction time of 0 minute. It was confirmed that the amount of theamplification product increased as the reaction time became longer from15 minutes to 30 or 60 minutes. However, the amount of the amplificationproduct as observed by electrophoresis was almost unchanged for areaction time of 60 minutes or longer, indicating that the amplificationin the reaction system used reached the plateau at about 60 minutes.

Example 2

(1) Preparation of RNA

An RNA used as a template in this Example was prepared from humancultured cell HT29 (ATCC HTB-38) (Dainippon Pharmaceutical) using TRIzolreagent (Life Technologies). The concentration of the resulting totalRNA was adjusted to 1 μg/μl. The OD260/OD280 value was 1.8, whichindicates the spectrophotometric purity of the RNA.

(2) Amplification Reaction

Bca BEST DNA polymerase, which has a reverse transcription activity anda DNA polymerase activity, as well as RNase H endonuclease were used todetermine if a cDNA is amplified from an RNA.

A reaction mixture having the composition as described in Example 2 wasprepared with the addition of 1 μg of the above-mentioned total RNA. Atargeted region encoding human transferrin receptor (GenBank accessionno. X01060) was amplified using the Primer Pair 2 in Example 1 asprimers.

The reaction mixture was incubated at 55° C. for 60 minutes, and thenheated at 90° C. for 2 minutes to inactivate the enzymes. When 8 μl ofthe reaction mixture was subjected to electrophoresis on 3% NuSieve 3:1agarose gel, an amplified fragment having an expected size of 56 bp wasobserved. Furthermore, Southern hybridization was carried out using aprobe having the targeted nucleotide sequence. A DNA probe having anucleotide sequence as shown in SEQ ID NO:12 of the Sequence Listinglabeled with biotin at the 5′-terminus was used to conduct Southernhybridization. As a result, the probe hybridized with theabove-mentioned amplified fragment, confirming that the targeted regionwas correctly amplified by the method of the present invention.

Example 3

(1) Synthesis of Primers

The amplification method of the present invention was examined using adouble-stranded DNA as a template. Primers used were synthesized using aDNA synthesizer (Applied Biosystems). Nucleotide sequences of primersare shown in SEQ ID NOS:13-22 of the Sequence Listing. The structures ofthe primers used in this Example are described below in detail. ThepUC19 DNA (Takara Shuzo) was used as a template for the Primer PairsA-F. The nucleotide sequence of pUC19 is available from a database(GenBank accession no. L09137). An amplified double-stranded DNAfragment was used as a template for the Primer Pair G. The fragment wasprepared from the human total RNA obtained in Example 2 using primershaving a sequence as shown in SEQ ID NOS:95 or 96 of the SequenceListing and TaKaRa RNA PCR Kit (AMV) Ver. 2.1 (Takara Shuzo) accordingto the attached standard protocol.

-   -   Primer Pair A (amplified fragment length: about 450 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO:13 or 14 of the Sequence Listing in which the first        and second bases from the 3′-terminus are ribonucleotides;    -   Primer Pair B (amplified fragment length: about 250 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO:13 or 15 of the Sequence Listing in which the first        and second bases from the 3′-terminus are ribonucleotides;    -   Primer Pair C (amplified fragment length: about 520 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO:13 or 16 of the Sequence Listing in which the first        and second bases from the 3′-terminus are ribonucleotides;    -   Primer Pair D (amplified fragment length: about 890 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO: 13 or 17 of the Sequence Listing in which the first        and second bases from the 3′-terminus are ribonucleotides;    -   Primer Pair E (amplified fragment length: about 130 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO:18 or 19 of the Sequence Listing in which the first to        third bases from the 3′-terminus are ribonucleotides;    -   Primer Pair F (amplified fragment length: about 220 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO:20 or 19 of the Sequence Listing in which the first to        third bases from the 3′-terminus are ribonucleotides; and    -   Primer Pair G (amplified fragment length: about 320 bp): A        combination of primers having a nucleotide sequence as shown in        SEQ ID NO:21 or 22 of the Sequence Listing in which the first to        third bases from the 3′-terminus are ribonucleotides.

(2) Amplification Reaction

A reaction mixture was prepared as follows.

35 mM potassium phosphate buffer (pH 7.5), 0.1 mg/ml bovine serumalbumin (BSA), 5% dimethyl sulfoxide, 1.4 mM each of dNTPs, 10 mMmagnesium chloride, 60 pmol each of the primers of one of the primerpairs as described above in (1), 100 ng of the pUC19 DNA as thetemplate, 5.5 U of Bca BEST DNA polymerase and 60 U of RNase H to afinal reaction volume of 50 μl.

The reaction conditions were as follows. The reaction mixture withoutthe DNA polymerase or RNase H was heat-denatured at 98° C. for 1 minute,and then cooled to 55° C. The DNA polymerase and RNase H were then addedthereto and the mixture was incubated at 55° C. for 60 minutes. Afterthe completion of the reaction, the mixture was heated at 90° C. for 2minutes to inactivate the enzymes. 8 μl of the reaction mixture was thensubjected to electrophoresis on 3% NuSieve 3:1 agarose gel.

As a result, it was confirmed that an amplified fragment of interest wasobtained using either of the Primer Pairs. Thus, it was confirmed that adouble-stranded DNA can be used as a template to conduct anamplification reaction in the amplification method of the presentinvention.

(3) Digestion of Amplification Product with Restriction Enzyme

Digestion of an amplified fragment obtained using the amplificationmethod of the present invention with a restriction enzyme was examined.The pUC19 plasmid DNA was used as a template DNA. pUC19 upper (2) NNprimer and pUC19 lower NN primer as shown in SEQ ID NOS:13 and 14 of theSequence Listing, respectively, were used. In the primers, the first andsecond bases from the 3′-terminus are ribonucleotides. The compositionof the reaction mixture was as follows.

Reaction Mixture A: 35 mM potassium phosphate buffer (pH 7.5), 10 mMmagnesium chloride, 1.4 mM each of dNTPs, 0.01% BSA, 5% DMSO, 2.7%glycerol, 100 pmol each of the pUC19 upper (2) NN primer and the pUC19lower NN primer, 500 ng of the pUC19 DNA and sterile distilled water toa reaction volume of 48 μl.

The reaction mixture was heat-denatured at 98° C. for 1 minute, and thencooled to 55° C. 60 U of E. coli RNase H and 5.5 U of Bca BEST were thenadded thereto to make the reaction volume to 50 μl. The reaction mixturewas incubated at 55° C. for 1 hour. After the completion of thereaction, the mixture was heated at 90° C. for 2 minutes to inactivatethe enzymes. The reaction mixture was subjected to electrophoresis on 3%agarose gel to purify the resulting amplification product. The recoveredamplification product was resupended in 100 μl of sterile distilledwater.

The thus obtained DNA solution was used for restriction enzymedigestion. Restriction enzymes used were AccII (Takara Shuzo) and BcnI(Takara Shuzo). The composition of the reaction mixture was as follows.

3 μl of the DNA solution, 1 μl of 10×AccII buffer or 10×BcnI bufferattached to each of the enzymes, 1 μl of the restriction enzyme AccII orBcnI and sterile distilled water to a reaction volume of 10 μl. Thereaction mixture was reacted at 37° C. for 30 minutes. 1.5 μl of 10×loading buffer was added thereto. 6 μl of the mixture was subjected toelectrophoresis on 3% NuSieve agarose gel.

As a result, restriction enzyme-digested DNA fragments of interest wereobtained using both of the restriction enzymes AccII and BcnI.

(4) Detection of Mutation

Detection of a mutation using the amplification method of the presentinvention was examined. pUC19 was used as a template. Nucleotidesequences of pUC19 upper (2) NN-U primer, pUC19 upper (2) NN-A primer,pUC19 upper (2) NN-C primer and pUC19 upper (2) NN-G primer are shown inSEQ ID NOS:23-26 of the Sequence Listing, respectively. Nucleotidesequences of pUC19 lower NN primer is shown in SEQ ID NOS:14 of theSequence Listing. Any one of these primers is a chimeric oligonucleotideprimer in which the first and second bases from the 3′-terminus areribonucleotides. The combinations of these primers were as follows.

-   -   Primer Pair 1: pUC19 upper (2) NN-U and pUC19 lower NN;    -   Primer Pair 2: pUC19 upper (2) NN-A and pUC19 lower NN;    -   Primer Pair 3: pUC19 upper (2) NN—C and pUC19 lower NN; and    -   Primer Pair 4: pUC19 upper (2) NN-G and pUC19 lower NN.

A reaction mixture was prepared as follows.

30 mM potassium phosphate buffer (pH 7.3), 0.01% bovine serum albumin(BSA), 5% DMSO, 1 mM each of dNTPs, 8 mM magnesium acetate, 60 pmol eachof the primers, 50 ng of the DNA as the template and sterile distilledwater to a reaction volume of 48 μl.

The reaction mixture was heat-denatured at 98° C. for 1 minute, and thencooled to 55° C. 5.5 U of Bca BEST DNA polymerase and 60 U of E. coliRNase H were then added thereto and the reaction mixture was incubatedat 55° C. for 60 minutes. The mixture was then heated at 90° C. for 2minutes to inactivate the enzymes. 8 μl of the reaction mixture wassubjected to electrophoresis on 4% NuSieve 3:1 agarose (Takara Shuzo)gel. As a result, an amplified fragment of about 450 bp of interest wasdetected only when the combination of the primers that included theprimer having a complementary base at the 3¹-terminus of pUC19 upper (2)NN was used. On the other hand, no amplified fragment was observed forthe combinations including the primer having a mismatched base at the3¹-terminus of pUC19 upper (2) NN.

Example 4

(1) Reaction in Microtube

Reaction volume for the amplification method of the present inventionwas examined. A region encoding human transferrin receptor was selectedas a region to be amplified. Primers having a sequence as shown in SEQID NO:27 or 28 of the Sequence Listing were used. In the primers, thefirst and second bases from the 3′-terminus are ribonucleotides. Afragment of about 750 bp amplified by RT-PCR was used as a template DNA.The reaction volume was adjusted to 50, 100, 300 or 500 μl. Thecomposition of the reaction mixture was as follows.

Reaction Mixture A: 10 μl of 5× specialized buffer (135 mM potassiumphosphate buffer (pH 7.5), 0.5 mg/ml BSA, 2.5% DMSO), 4 μl of 100 mMmagnesium acetate, 5 μl of 10 mM dNTPs, 10 μl of 10 μM ATP, 1 μl of BcaBEST DNA polymerase (22 U/μl), 1 μl of RNase H (60 U/μl) and steriledistilled water to 39 μl.

Reaction Mixture B: 3 μl each of 20 μM human transferrin receptor Sprimer (SEQ ID NO:27) and 20 μM human transferrin receptor A primer (SEQID NO:28), about 100 ng of the DNA as the template and sterile distilledwater to 11 μl. If the volume became 50 μl or more, it was scaled up tohave the above-mentioned composition.

For an amplification reaction, the Reaction Mixture B was treated at 98°C. for 2 minutes, and then incubated at 55° C. for 3 minutes. TheReaction Mixture B was added to the Reaction Mixture A which had beenpre-incubated in a 1500-μl microtube at 55° C. After mixing, thereaction mixture was incubated at 55° C. for 1 hour. After thecompletion of the reaction, the mixture was transferred to an ice bath.8 μl of the reaction mixture was subjected to electrophoresis on 3%agarose gel.

As a result, a fragment of about 300 bp of interest was efficientlyamplified using each of the reaction volumes. In addition, it wasconfirmed that an amplified fragment of interest can be obtained withouta problem using a PCR-amplified fragment as a template DNA.

(2) Reaction in Petri Dish

Use of a Petri dish for preventing the heterogeneous temperature in areaction mixture due to increased reaction volume was examined. A regionencoding human transferrin receptor was selected as a region to beamplified. Primers having a sequence as shown in SEQ ID NO:12 or 13 ofthe Sequence Listing were used. In the primers, the first and secondbases from the 3′-terminus are ribonucleotides. A fragment of about 750bp amplified by RT-PCR was used as a template DNA. The reaction volumewas adjusted to 10 ml. The composition of the reaction mixture was asfollows.

Reaction Mixture A: 2000 μl of 5× specialized buffer (135 mM potassiumphosphate buffer (pH 7.5), 0.5 mg/ml BSA, 2.5% DMSO), 800 μl of 100 mMmagnesium acetate, 1000 μl of 10 mM dNTPs and sterile distilled water to9.1 ml.

Reaction Mixture B: 200 μl each of 60 μM human transferrin receptor Sprimer (SEQ ID NO:12) and 60 μM human transferrin receptor primer (SEQID NO:13), about 10 μg of the DNA as the template and sterile distilledwater to 500 μl.

Reaction Mixture C: 200 μl of Bca BEST DNA polymerase (22 U/μl) and 200μl of RNase H (60 U/μl)

For an amplification reaction, the Reaction Mixture B was treated at 98°C. for 1 minute, and then incubated at 55° C. for 3 minutes. TheReaction Mixture B was added to the Reaction Mixture A which had beenpre-incubated in a 60-mm (diameter) plastic Petri dish at 55° C. TheReaction Mixture C was further added thereto. After mixing, the reactionmixture was incubated at 55° C. for 1 hour. After the completion of thereaction, the reaction mixture was transferred to an ice bath. 8 μl ofthe reaction mixture was then subjected to electrophoresis on 3% agarosegel.

As a result, a fragment of about 300 bp of interest was efficientlyamplified even if the reaction volume of 10 ml was used. In addition, itwas confirmed that an amplified fragment of interest can be obtainedwithout a problem using a PCR-amplified fragment as a template DNA.Thus, it was confirmed that the method of the present invention can bemore preferably used for making a DNA chip, which requires a largeamount of a DNA fragment, as compared to the conventional PCR method.

Example 5

(1) Relationship Between Type of Buffer and Amount of RNase H Used

The relationship between the type of buffer and the amount of RNase Hused was examined. Plasmid DNAs, in which a fragment of 249 bp or 911 bpwas cloned into the pUC19 vector (designated as pUC19-249 and pUC19-911)were used as templates. Chimeric oligonucleotide primers, in which thefirst to third bases from the 3′-terminus of MF2N3 (24) primer or MR1N3(24) primer having a sequence as shown in SEQ ID NO:29 or 30 of theSequence Listing are ribonucleotides, were used as primers. By using thecombination of these primers, amplified fragments of about 450 bp andabout 1100 bp are obtained for pUC19-249 and pUC19-911, respectively.

A tris-hydrochloride buffer, a potassium phosphate buffer and Tricinebuffer were selected as buffer systems to be examined. The amounts ofRNase H examined were no addition and a final concentration ranging from0.3 to 1.2 U/μl. The tris-hydrochloride buffer system was prepared asdescribed in Example 1 (2), except that 10 ng of pUC19-249 or 200 ng ofpUC19-911, 60 pmol each of the primers and 11 U/50 μl reaction volume ofBca BEST DNA polymerase were used. The potassium phosphate buffer systemwas prepared to have a similar composition. The Tricine buffer systemwas prepared to contain the following at the indicated finalconcentration: 34 mM Tricine buffer (pH 8.7), 10 mM potassium chloride,10 mM ammonium sulfate, 0.01% BSA, 1% DMSO, 4 mM magnesium acetate and0.5 mM each of dNTPs. 10 ng/50 μl reaction volume of the pUC19-249plasmid or 200 ng/50 μl reaction volume of the pUC19-911 plasmid, 60pmol/50 μl reaction volume each of primers, RNase H at a predeterminedconcentration and 11 U/50 μl reaction volume of Bca BEST DNA polymerasewere added to the buffer system.

For an amplification reaction, a mixture of pUC19-249 or pUC19-911 as atemplate and the respective primers was heat-denatured at 98° C. for 1minute, and then cooled to 55° C. A mixture of the remaining reactioncomponents was added thereto. The mixture was reacted at 55° C. for 60minutes. After the completion of the reaction, the mixture was cooled to4° C. and 1/10 volume of 0.5 M EDTA was added thereto to terminate thereaction. 3 μl of the reaction mixture was subjected to electrophoresison 3% NuSieve 3:1 agarose (Takara Shuzo) gel.

As a result, when pUC19-249 was used as a template, increase inamplification efficiency was observed depending on the buffer systemused in the following order: tris-hydrochloride<potassiumphosphate<Tricine. When pUC19-911 was used as a template, increase inamplification efficiency was observed depending on the buffer systemused in the following order: tris-hydrochloride<Tricine<potassiumphosphate. The use of RNase H at a final concentration ranging from 0.3to 1.2 U/μl resulted in the amplified fragment of interest, although noamplified fragment of interest was observed for no addition.

(2) Examination of Amount of Primer

The effect of the amount of a primer used on the amplification method ofthe present invention was examined. A reaction mixture system having acomposition in which pUC19-249 was used as a template among thecompositions as described above in (1) was used. 60 U/50 μl reactionvolume of RNase H was used for the potassium phosphate buffer system,whereas 30 U/50 μl reaction volume of RNase H was used for thetris-hydrochloride or Tricine buffer system. The examined concentrationof the primer ranged from 10 to 100 pmol/50 μl. Reaction conditions andconfirmation of amplification were as described above in (1).

As a result, an amplified fragment of interest was observed using eachof the reaction buffer systems containing the primer at a concentrationranging from 10 to 100 pmol/50 μl.

(3) Effect of pH of Reaction Buffer

The effect of the pH of a reaction mixture on the amplification methodof the present invention was examined. The composition of the reactionmixture was as described above in (2). The pH examined were 7.0-8.0 forthe potassium phosphate buffer system, 7.5-9.2 for the Tricine buffersystem, and 7.5-9.0 for the tris-hydrochloride buffer system. Reactionconditions and confirmation of amplification were as described above in(1).

As a result, an amplified fragment of interest was observed at pH withinthe range used for the respective buffer systems.

(4) Effect of Additive

The effect of addition of dimethyl sulfoxide (DMSO) was examined usingthe reaction mixture composition of the phosphate buffer system (pH 7.5)as described above in (3). Additionally, the effect of addition of apolyamine was also examined. The examined amount of added DMSO rangedfrom no addition to 10%. On the other hand, spermine tetrahydrochloride(Sigma), spermidine trihydrochloride (Sigma), acetylputrescine (NacalaiTesque), putrescine dihydrochloride (Nacalai Tesque), trimethylenediamine (Nacalai Tesque), propylenediamine (Nacalai Tesque) anddiaminomethane dihydrochloride (Nacalai Tesque) were used as apolyamine. The amounts of propylenediamine and trimethylene diamineadded were within the range between no addition and 2%. Otherpolyaminies were used within the range between no addition and 5 mM.Reaction conditions and confirmation of amplification were as describedabove in (1).

As a result, a DNA fragment of interest was efficiently amplified usingthe additive at a concentration within the indicated range: no additionto 5% of DMSO; no addition to 200 μM of spermine tetrahydrochloride orspermidine; 40 μM to 40 mM of acetylputrescine or putrescinedihydrochloride; 0.002% to 0.02% of trimethylene diamine; 0.0001% to0.01% of propylenediamine; and 0.1 μM to 10 μM of diaminomethanedihydrochloride.

(5) Examination of Type of Magnesium Salt

The effect of the type of a magnesium salt on the amplification methodof the present invention was examined. The pUC19 DNA was used as atemplate. pUC19 upper NN 249 primer and pUC19 lower NN primer havingsequences as shown in SEQ ID NOS:31 and 14 of the Sequence Listing,respectively, were used as primers. An amplified fragment of about 225bp is obtained using a pair of these primers. Magnesium chloride,magnesium acetate and magnesium sulfate were used as magnesium salts.The composition of the reaction mixture was as follows.

35 mM potassium phosphate buffer (pH 7.3), 8 mM (final concentration)magnesium chloride, magnesium acetate or magnesium sulfate, 1.0 mM(final concentration) each of dNTPs, 50 ng of the pUC19 DNA, 60 pmoleach of the primers, 60 U of RNase H, 5.5 U of Bca BEST DNA polymeraseand sterile distilled water to a reaction volume of 50 μl. Reactionconditions and confirmation of amplification were as described above in(3).

As a result, an amplified fragment of interest was observed using eachof the magnesium salts.

(6) Examination of Concentrations of Magnesium and dNTPs

The effects of the concentrations of magnesium and dNTPs on theamplification method of the present invention were examined. Thecomposition of the reaction mixture was as described above in (5),except that 25 ng of the pUC19 DNA, and magnesium and dNTPs at variousconcentrations were used. Reaction conditions and confirmation ofamplification were as described above in (1).

In a reaction system in which the final concentration of each of dNTPswas fixed at 1 mM, an amplified fragment of interest was obtained when afinal magnesium concentration ranging from 6 mM to 10 mM was used. In areaction system in which the final magnesium concentration of was fixedat 8 mM, an amplified fragment of interest was obtained when a finalconcentration of each of dNTPs ranging from 0.6 mM to 1.2 mM was used.Furthermore, in a reaction system in which the final concentration ofeach of dNTPs was fixed at 0.5 mM, an amplified fragment of interest wasobtained when a final magnesium concentration ranging from 2 mM to 6 mMwas used. In a reaction system in which the final magnesiumconcentration was fixed at 4 mM, an amplified fragment of interest wasobtained when a final concentration of each of dNTPs ranging from 0.2 mMto 0.8 mM was used.

(7) Examination of Change in Concentration of Potassium Phosphate Bufferor Tricine Buffer and Reactivity

The effect of the concentration of the potassium phosphate buffer or theTricine buffer on the amplification method of the present invention wasexamined. The composition of the reaction mixture was as described abovein (1) for a case where pUC19-249 was used as a template, except that apotassium phosphate buffer at a final concentration of 20-50 mM or aTricine buffer at a final concentration of 22-46 mM was used.

As a result, an amplified fragment of interest was obtained when thepotassium phosphate buffer at a final concentration ranging from 20 to50 mM or the Tricine buffer at a final concentration ranging from 22 to46 mM was used.

(8) Examination of Concentration of Bca BEST DNA Polymerase

The effect of the concentration of Bca BEST DNA polymerase on theamplification method of the present invention was examined. Thecomposition of the reaction mixture was as described above in (1) for acase where pUC19-249 was used as a template, except that a potassiumphosphate buffer system or a Tricine buffer system and Bca BEST DNApolymerase at a concentration within a range of 1-22 U/50 μl reactionvolume was used. Reaction conditions and confirmation of amplificationwere as described above in (1).

As a result, an amplified fragment of interest was obtained when BcaBEST DNA polymerase was used at a concentration within a range of 1-22U/50 μl.

Example 6

Comparison with the PCR Method

The amplification method of the present invention was compared with thePCR method. Ones in which a DNA fragment of about 150 bp or about 250 bpis inserted into a multi-cloning site in the pUC19 plasmid DNA were usedas templates. The templates were prepared as follows.

-   -   pUC19 upper 150 PCR primer, pUC19 upper 249 PCR primer and pUC19        lower PCR primer, which have sequences as shown in SEQ ID        NOS:32, 33 and 34 of the Sequence Listing, respectively, were        used to conduct a PCR reaction using 100 μg of the pUC19 plasmid        DNA as a template. An amplified fragment of about 150 bp was        obtained by using a combination of the pUC19 upper 150 primer        and the pUC19 lower NN primer. An amplified fragment of about        250 bp was obtained by using a combination of the pUC19 upper        249 primer and the pUC19 lower NN primer. Each of these        amplified fragments was purified using Microcon-100, blunt-ended        using DNA blunting kit (Takara Shuzo), and then subcloned into a        HincII site in the pUC19 plasmid. Plasmids into which one of the        amplified fragments is inserted were used to transform E. coli        JM109. The resulting transformants were cultivated and plasmids        with inserted DNA were purified from the cells using QIAGEN        plasmid mini kit (Qiagen). The plasmids with inserted DNA were        used as templates.

The sequences of the primers used in this Example are shown in SEQ IDNOS:35 and 36 of the Sequence Listing. Primers in which the first tothird bases from the 3′-terminus are ribonucleotides were used for theamplification method of the present invention. The composition of thereaction mixture was as follows.

27 mM phosphate buffer (pH 7.3), 0.01% bovine serum albumin (BSA), 5%DMSO, 1 mM each of dNTPs, 8 mM magnesium acetate, 60 pmol each of theprimers, 1 ng of the DNA as the template and sterile distilled water toa reaction volume of 48 μl.

The reaction mixture was heat-denatured at 98° C. for 1 minute, and thencooled to 55° C. 5.5 U of Bca BEST DNA polymerase and 60 U of E. coliRNase H were added thereto, and the mixture was incubated at 55° C. for60 minutes. Thereafter, the mixture was heated at 90° C. for 2 minutesto inactivate the enzymes. 3 μl of the reaction mixture was subjected toelectrophoresis on 4% NuSieve 3:1 agarose (Takara Shuzo) gel.

On the other hand, amplification using the PCR method was conducted as acontrol. PCR Amplification kit (Takara Shuzo), 10 pmol each of primershaving a sequence as shown in SEQ ID NO:37 or 38 of the SequenceListing, 1 ng of the DNA as the template and sterile distilled water toa reaction volume of 50 μl were used for the reaction. The reactionconditions were 25 cycles of 94° C. for 30 seconds, 55° C. for 30seconds and 72° C. for 40 seconds. After the completion of the reaction,3 μl of the reaction mixture was subjected to electrophoresis on 4%NuSieve 3:1 agarose (Takara Shuzo) gel.

As a result, more amount of a fragment of interest was amplified fromeach of the plasmids having an insert of 150 bp or 249 bp as a templatein the amplification method of the present invention as compared withthe PCR method. 20 μl of the reaction mixture was purified usingMicrocon-100, and the amount of the amplification product was quantifiedusing Beckman DU-640 spectrophotometer (Beckman) in order to expressnumerically the amount of the amplification product. The amount of thefragment amplified from the plasmid having an insert of 150 bp as thetemplate in the amplification method of the present invention wasconfirmed to be about 60-fold higher than that in the PCR method. Theamount of the fragment amplified from the plasmid having an insert of250 bp as the template in the amplification method of the presentinvention was confirmed to be about 40-fold higher than that in the PCRmethods. Based on these results, it was confirmed that the method of thepresent invention can be more preferably used for making a DNA chip, forwhich a large amount of a DNA fragment is required, as compared with theconventional PCR method.

Example 7

(1) Preparation of RNA Probe

A method for detecting an amplified fragment obtained by theamplification method of the present invention was examined. A probe fordetection composed of ribonucleotides in which two different fluorescentsubstances are attached to the ribonucleotides on both ends of the probewas prepared. The RNA probe for detection was synthesized using a DNAsynthesizer (Applied Biosystems). The nucleotide sequence of the probeis shown in SEQ ID NO:39 of the Sequence Listing. 6-FAM (Glen Research)and TAMRA (Glen Research) were used as fluorescent substances to labelthe probe at the 5′-terminus and the 3′-terminus, respectively.

(2) Amplification Reaction and Detection

0.1 or 1 ng of the pUC19 DNA was used as a template. pUC19 upper 150 2Nprimer and pUC19 lower 542 2N primer having sequences as shown in SEQ IDNOS:40 and 16 of the Sequence Listing, respectively, in which the firstand second bases from the 3′-terminus of the primer are ribonucleotideswere used as primers.

The composition of the reaction mixture was as follows.

27 mM phosphate buffer (pH 7.3), 0.01% BSA, 5% DMSO, 1 mM each of dNTPs,8 mM magnesium acetate, 60 pmol each of the primers, 0.1 or 1 ng of theDNA as the template, 0.1 μg of the RNA probe and sterile distilled waterto a reaction volume of 48 μl. One without the DNA as the template wasalso prepared as a control.

The reaction mixture was heat-denatured at 98° C. for 1 minute, and thencooled to 55° C. 22 U of Bca BEST DNA polymerase or sterile water, and60 U of E. coli RNase H were added thereto, and the mixture wasincubated at 55° C. for 60 minutes. Thereafter, 5 μl of 10% sodiumdodecyl sulfate (SDS; Nacalai Tesque) was added to the mixture toinactivate the enzymes. 50 μl of the reaction mixture was diluted withan equal volume of sterile water and transferred to a microplate. Animage analyzer FM BIO II Multi-View (Takara Shuzo) was used fordetection at an excitation wavelength of 505 nm.

As a result, no fluorescent signal was detected using either of thetemplates when Bca BEST DNA polymerase was not added. Also, nofluorescent signal was detected for the reaction mixture containing BcaBEST DNA polymerase when the DNA as the template was not added. On theother hand, a fluorescent signal was detected when either 0.1 or 1 ng ofthe DNA as the template was added. An amplified fragment of about 190 bpof interest was also observed by electrophoresis on 3% agarose gelcontaining 0.00003% ethiduim bromide only when 0.1 or 1 ng of the DNA asthe template was added in the presence of Bca BEST DNA polymerase. Thatis, the same results were obtained by the detection method using an RNAprobe and the conventional electrophoretic detection method. Thus, amethod for detecting an amplified fragment obtained by the amplificationmethod of the present invention using an RNA probe was established.

Example 8

Use of a primer composed of deoxyribonucleotides as one of the twoprimers in the method of the present invention was examined. MR1N3 (30)having a sequence as shown in SEQ ID NO:41 of the Sequence Listing andM4 primer (Takara Shuzo) having a sequence as shown in SEQ ID NO:42 ofthe Sequence Listing were used as primers. In the MR1N3 primer, thefirst to third bases from the 3′-terminus are ribonucleotides. Thecomposition of the reaction mixture was as follows.

27 mM phosphate buffer (pH 7.3), 0.01% bovine serum albumin (BSA), 5%DMSO, 1 mM each of dNTPs, 8 mM magnesium acetate, 30 pmol each of theprimers, 1 ng of the DNA as the template and sterile distilled water toa reaction volume of 24 μl.

The reaction mixture was heat-denatured at 98° C. for 2 minutes, andthen cooled to 55° C. 11 U of Bca BEST DNA polymerase and 30 U of E.coli RNase H were added thereto to make the reaction volume to 25 μl.The reaction mixture was incubated at 55° C. for 60 minutes. Thereafter,the mixture was heated at 90° C. for 2 minutes to inactivate theenzymes. 5 μl of the reaction mixture was subjected to electrophoresison 4% NuSieve 3:1 agarose gel. As a result, an amplified fragment ofinterest was observed.

Example 9

The method of the present invention was used to detect hemorrhagic E.coli O-157.

Sequences of the primers used in this Example are shown in SEQ IDNOS:43-46 of the Sequence Listing. In the primers, the first, second andthe third bases from the 3′-terminus are ribonucleotides. A combinationof primers having a sequence of SEQ ID NO:40 or 41, and a combination ofprimers having a sequence of SEQ ID NO:42 or 43 were constructed fordetecting a sequence encoding vero toxin 1 or vero toxin 2 of O-157according to the description of Rinsho To Biseibutsu (ClinicalMicrobiology), 18(4):507-513 (1991). Primers in which the first to thirdbases from the 3′-terminus are ribonucleotides were used for theamplification method of the present invention. A heat-extract preparedby harvesting a culture of hemorrhagic E. coli O-157 (ATCC accession no.43895), suspending it in sterile water at an appropriate cell densityand treating it at 98° C. for 10 minutes was used as a template. Thecomposition of the reaction mixture was as follows.

27 mM phosphate buffer (pH 7.3), 0.01% bovine serum albumin (BSA), 5%DMSO, 1 mM each of dNTPs, 8 mM magnesium acetate, 60 pmol each of theprimers, the DNA as the template (the heat-extract) corresponding to10⁴-10⁶ cells and sterile distilled water to a reaction volume of 48 μl.

The reaction mixture was heat-denatured at 98° C. for 1 minute, and thencooled to 55° C. 5.5 U of Bca BEST DNA polymerase and 60 U of E. coliRNase H were added thereto. The reaction mixture was incubated at 55° C.for 60 minutes. Thereafter, the mixture was heated at 90° C. for 2minutes to inactivate the enzymes. 3 μl of the reaction mixture wassubjected to electrophoresis on 4% NuSieve 3:1 agarose (Takara Shuzo)gel.

As a result, O-157 vero toxin 1 and 2 could be detected using either oneof the primer pairs and the DNA as the template corresponding to 10⁴cells, confirming that the method of the present invention can beutilized as a method for detecting a virulent bacterium.

Example 10

Amplification of a long-chain DNA fragment by the method of the presentinvention was examined. A double-stranded DNA as a template was preparedas follows. First, a library was constructed from mRNA derived from anormal gastric tissue using Uni-ZAP XR vector (Stratagene) according toa conventional method. The library was screened to select clones havingan insert of about 2.1 kbp or about 4.3 kbp. The clones were used toobtain pBluescript SK (−) phage vectors by in vitro excision. Amplifiedfragments of about 2.2 kbp and about 4.4 kbp were obtained using theplasmids as templates, MCR-F primer and MCR-R primer having sequences asshown in SEQ ID NOS:47 and 48 of the Sequence Listing, respectively, andPCR Amplification kit (Takara Shuzo). These PCR fragments were used astemplates for the amplification method of the present invention. MF2N3(24) primer and MR1N3 (24) primer having sequences as shown in SEQ IDNOS:49 and 50 of the Sequence Listing, respectively, in which the firstto third bases from the 3′-terminus are ribonucleotides were used asprimers. The composition of the reaction mixture was as follows.

28 mM phosphate buffer (pH 7.5), 0.01% bovine serum albumin (BSA), 1%DMSO, 0.5 mM each of dNTPs, 4 mM magnesium acetate, 30 pmol each of theprimers, 0.2 mM putrescine and sterile distilled water to 24.25 μl. Thereaction mixture was treated at 92° C. for 2 minutes, and then cooled to55° C. 30 U of RNase H and 5.5 U of Bca BEST DNA polymerase were addedthereto to make the reaction volume to 25 μl. The reaction mixture wasincubated for 1 hour. After the completion of the reaction, the mixturewas cooled at 4° C., and 2.5 μl of a 0.5 M EDTA solution added theretoto terminate the reaction. 5 μl of the mixture was subjected toelectrophoresis on 1% agarose gel.

As a result, an amplified fragment of about 2.2 kbp or about 4.4 kbp wasobtained by the method of the present invention, confirming that themethod of the present invention can be used to amplify a long-chain DNAfragment.

Example 11

A DNA microarray onto which a λ DNA fragment of about 400 bp amplifiedby the amplification method of the present invention and λ DNA fragmentsof 300 bp and 1000 bp amplified by PCR were spotted was produced. Thenucleotide sequence of the λ DNA is available from GenBank accessionnos. V00636, J02459, M17233 and X00906. A reaction mixture for theamplification method of the present invention was prepared as follows.

34 mM Tricine-hydrochloride buffer (pH 8.7), 10 mM potassium chloride,10 mM ammonium sulfate, 0.01% bovine serum albumin (BSA), 1% dimethylsulfoxide, 4 mM magnesium acetate, 0.5 mM each of dNTPs, 500 pmol eachof the primers, 100 ng of the PCR amplification product as the template,110 U of Bca BEST DNA polymerase and 300 U of cloned RNase H in a finalreaction volume of 500 μl. The reaction mixture was mixed tohomogeneity, incubated at 55° C. for 60 minutes, and then heated at 90°C. for 2 minutes to inactivate the enzymes. This solution was used inthe subsequent steps. The spotted DNA fragments were as follows.

1. Sample: A PCR amplification product (300 bp) obtained by using a λDNA as a template and a combination of primers having a sequence asshown in SEQ ID NO:51 or 52 of the Sequence Listing was subcloned intothe pUC19 vector. The subcloned product was then PCR-amplified usingMCR-F primer and MCR-R primer as shown in example 10. The thus obtainedproduct as a template and chimeric oligonucleotide primers having asequence as shown in SEQ ID NO:53 or 54 of the Sequence Listing, inwhich the first and second bases from the 3′-terminus of the primer areribonucleotides, were used to amplify a product of about 400 bp by theamplification method of the present invention to obtain the Sample. FiveDNA solutions, i.e., the reaction mixture at its original concentrationor 2-, 4-, 8- or 16-fold dilutions of the reaction mixture with acarbonate buffer (a carbonate buffer at a concentration of 50 mM wereused for dilution in each case) were used for spotting.

2. Sample: The DNA fragment amplified in 1 above was treated withMicrocon-100 (Takara Shuzo). Then, five DNA solutions were prepared byadjusting the concentrations to 0.125 μg/μl, 0.25 μg/μl, 0.5 μg/μl, 1.0μg/μl and 2.0 μg/μl with the 50 mM carbonate buffer.

3. Positive Control: A PCR amplification product (300 bp) amplified in 1above was treated with Microcon-100. Then, five DNA solutions wereprepared by adjusting the concentrations to 0.125 μg/μl, 0.25 μg/μl, 0.5μg/μl, 1.0 μg/μl and 2.0 μg/μl with the 50 mM carbonate buffer.

4. Positive Control: A PCR amplification product (1000 bp) obtained byusing the λ DNA as a template and a combination of primers having asequences as shown in SEQ ID NO:55 or 56 of the Sequence Listing wastreated with Microcon-100. Then, four DNA solutions were prepared byadjusting the concentrations to 0.125 μg/μl, 0.25 μg/μl, 0.5 μg/μl and1.0 μg/μl with the 50 mM carbonate buffer.

5. Negative Control: A PCR amplification product (300 bp) obtained byusing the λ DNA as a template and a combination of primers having asequence as shown in SEQ ID NO:55 or 57 of the Sequence Listing wassubcloned into the pUC19 vector. The subcloned product was thenPCR-amplified using primers having a sequence as shown in SEQ ID NO:47or 48 of the Sequence Listing. The thus obtained product as a templateand primers having a sequence as shown in SEQ ID NO:53 or 54 of theSequence Listing as primers were used to amplify a product of about 400bp by the amplification method of the present invention to obtain theNegative Control. Five DNA solutions, i.e., the reaction mixture at itsoriginal concentration or 2-, 4-, 8- or 16-fold dilutions of thereaction mixture with a carbonate buffer (a carbonate buffer at aconcentration of 50 mM were used for dilution in each case) were usedfor spotting.

6. Negative Control: The DNA fragment obtained in 5 above was treatedwith Microcon-100. Then, five DNA solutions were prepared by adjustingthe concentrations to 0.125 μg/μl, 0.25 μg/μl, 0.5 μg/μl, 1.0 μg/μl and2.0 μg/μl with the 50 mM carbonate buffer.

The respective DNA solutions thus prepared were spotted onto a slideglass to which amino groups had been introduced (Matsunami Glass) usingan equipment for making DNA chips (Genetic Microsystems (GMS)), and wereimmobilized using UV irradiation. The slide was washed with 0.2% SDSfollowed by distilled water, dried, and then used as a DNA array.

A PCR amplification product (300 bp) amplified in 1 above was labeledwith Cy5 using Label IT Cy5^(R) Labeling Kit (Takara Shuzo) for use as aprobe. Hybridization was carried out using a prehybridization solutionand a hybridization solution as described in the instructions attachedto IntelliGene (Takara Shuzo). First, the DNA array was subjected toprehybridization at room temperature for 2 hours. The hybridizationsolution containing the denatured Cy5-labeled probe was dripped onto theDNA array. A cover glass was mounted thereon. The sides of the coverglass were sealed with a film. The sealed DNA array was incubated at 65°C. for 13 hours. After the cover glass was removed, the DNA array waswashed in 2×SSC at 65° C. for 5 minutes, in a solution containing0.2×SSC and 0.1% SDS at 65° C. for 5 minutes, and finally in 0.2×SSC atroom temperature for 5 minutes, and air-dried. The DNA array was thensubjected to a microarray scanner (GMS) to analyze the fluorescentsignals from the respective spots.

As a result, a fluorescent signal was observed at each of the positionsonto which fragments amplified by the PCR method (the Positive Controlsas described above in 3 and 4) and the method of the present invention(the Samples as described above in 1 and 2) were spotted. Theintensities of the signals were as follows: the Sample 2>the PositiveControl 4>the Sample 1>the Positive Control 3. On the other hand, nosignal was observed at all for the Negative Controls 5 and 6. From theseresults, it was confirmed that an unpurified or purified DNA fragmentamplified by the method of the present invention can be preferably usedas a DNA fragment to be immobilized to make a DNA chip.

Example 12

(1) The designing of a primer used in the method of the presentinvention in which a PCR-amplified fragment was used as a template wasexamined. First, R1-S1 primer, R1-A3 primer, R2-S1 primer, R2-A3 primer,R3-S1 primer and R3-A3 primer having sequences as shown in SEQ IDNOS:58-63 of the Sequence Listing respectively, were synthesizedaccording to a conventional method. The structures of the respectiveprimers are as follows.

-   -   (i) R1-S1 primer: From the 5′-terminus, 7 bases of a spacer        sequence, 17 bases of a M13RV sequence (or RV sequence; the        nucleotide sequence of M13RV primer (Takara Shuzo)) and 20 bases        of a sense primer sequence for λ DNA-specific PCR;    -   (ii) R1-A3 primer: From the 5′-terminus, 7 bases of a spacer        sequence, 17 bases of the M13RV sequence and 20 bases of an        antisense primer sequence for λ DNA-specific PCR;    -   (iii) R2-S1 primer: From the 5′-terminus, 25 bases of a spacer        sequence, 17 bases of the M13RV sequence and 20 bases of a sense        primer sequence for λ DNA-specific PCR;    -   (iv) R2-A3 primer: From the 5′-terminus, 25 bases of a spacer        sequence, 17 bases of the M13RV sequence and 20 bases of an        antisense primer sequence for λ DNA-specific PCR;    -   (v) R3-S1 primer: From the 5′-terminus, 58 bases of a spacer        sequence, 17 bases of the M13RV sequence and 20 bases of a sense        primer sequence for λ DNA-specific PCR; and    -   (vi) R3-A3 primer: From the 5′-terminus, 58 bases of a spacer        sequence, 17 bases of the M13RV sequence and 20 bases of an        antisense primer sequence for λ DNA-specific PCR.

M13RV 20mer has a sequence of a total of 20 bases consisting of 17 basesof the M13RV sequence and 3 bases at the 5′-terminus. Therefore, whenM13RV 20 mer is used in the method of the present invention, the lengthsof the spacer sequences in the above-mentioned primers become 4 bases,22 bases and 55 bases, respectively. Primers without a spacer sequencewere also made as controls for the above-mentioned primers.

For example, when the primer pair, R1-S1 primer/R1-A3 primer, is used,an amplified fragment of 348 bp is obtained. 7 bases on both ends of theamplified fragment correspond to the spacer portions. The RV sequencesare located inside the spacer portions. The λ DNA sequences are locatedinside the RV sequences.

Similarly, when the primer pair, R2-S1 primer/R2-A3 primer, is used, anamplified fragment of 384 bp in which 25 bases on both ends of theamplified fragment correspond to the spacer portions is obtained. Inaddition, when the primer pair, R3-S1 primer/R3-A3 primer, is used, anamplified fragment of 450 bp in which 58 bases on both ends of theamplified fragment correspond to the spacer portions is obtained. On theother hand, a fragment amplified using control primers has no spacerportion. These PCR-amplified fragments were used as templates for thefollowing examination.

One of two primers, M13RV-2N 17mer primer or M13RV-2N 20mer having asequence as shown in SEQ ID NO:64 or 65 of the Sequence Listing was usedin this Example. In the primers, the first and second bases from the3′-terminus are ribonucleotides. The reaction was carried out asfollows. 5 μl of a mixture of 20 μM of the primer, about 20 ng of thetemplate and 0.01% propylenediamine was denatured at 98° C. for 2minutes, and then cooled to 55° C. Thereafter, 34 mM Tricine buffer (pH8.7), 10 mM potassium chloride, 10 mM ammonium sulfate, 0.01% BSA, 1%DMSO, 4 mM magnesium acetate, 0.5 mM each of dNTPs, 1 U of Bca BEST DNApolymerase and 15 U of RNase H were added thereto to make the finalreaction volume to 25 μl. The reaction mixture was incubated at 55° C.for 1 hour. After the completion of the reaction, the mixture was cooledto 4° C., and then 2.5 μl of a 0.5 M EDTA solution was added thereto toterminate the reaction. 3 μl of the reaction mixture was subjected toelectrophoresis on 3% NuSieve 3:1 agarose (Takara Shuzo) gel. As aresult, when M13RV-2N 17mer was used, increase in amplificationefficiency was observed depending on the length of the spacer sequencein the following order: 25mer>7mer>58mer>no spacer sequence. WhenM13RV-2N 20mer was used, increase in amplification efficiency wasobserved depending on the length of the spacer sequence in the followingorder: 22mer>4mer>55mer>no spacer sequence. Furthermore, when the M13RVsequences in the primers described above in (i) to (vi) were replaced byM13M4 sequences, a similar tendency was observed for the relationshipbetween the spacer sequence and the amplification efficiency. Thus, itwas confirmed that, when a linear DNA fragment such as a PCR-amplifiedfragment is used as a template, the designing of primers used in themethod of the present invention to generate a spacer sequence (portion)leads to an increased amplification efficiency.

(2) Amplification of a template having a high GC content in the methodfor amplifying a nucleotide sequence using an elevated reactiontemperature was examined. First, primers having a sequence as shown inSEQ ID NO:66 or 67 of the Sequence Listing for PCR amplification of a307-bp region (GC content: 62.5%) of CDC2-related protein kinase PISSLRE gene (GenBank accession no. AA789328) were produced. In addition,primers having a sequence as shown in SEQ ID NO:68 or 69 of the SequenceListing for PCR amplification of a 284-bp region (GC content: 61.3%) ofType II cytoskeltal 1 keratin gene (GenBank accession no. AA706022) wereproduced. PCR amplification was carried out using these primers andcommercially available DNA fragments (Research Genetics) as templates.The respective PCR-amplified fragments obtained by using theabove-mentioned primer pairs have spacer sequences and the M13RVsequences on both ends. The fragments were used as templates for thepresent invention.

M13RV-2N 17mer primer having a sequence as shown in SEQ ID NO:64 of theSequence Listing or M13RV-2N 20mer primer having a sequence as shown inSEQ ID NO:65 of the Sequence Listing was used in this Example. In theprimers, the first and second bases from the 3′-terminus areribonucleotides. The reaction was carried out as follows. μl of amixture of 100 pmol of the primer, 20 ng of the template and 0.01%propylenediamine was denatured at 980 C for 2 minutes, and then cooledto 550 C or 600 C. Thereafter, 34 mM Tricine buffer (pH 8.7), 10 mM KCl,10 mM ammonium sulfate, 0.01% BSA, 1% DMSO, 4 mM magnesium acetate, 0.5mM each of dNTPs, 11 U of Bca BEST DNA polymerase and 30 U of RNase Hwere added thereto to make the final reaction volume to 50 μl. Thereaction mixture was incubated at 55° C. or 60° C. for 1 hour. After thecompletion of the reaction, the mixture was cooled to 4° C. 3 μl of thereaction mixture was subjected to electrophoresis on 3% agarose gel. Theresults are shown in Table 1 below. TABLE 1 Gene and results ofamplification Reaction Primers Type II temperature used CDC2-relatedcytoskeltal 55° C. M13RV-2N 17mer ++ ++ M13RV-2N 20mer ++ ++ 60° C.M13RV-2N 17mer + + M13RV-2N 20mer ++++ +++++ to ++++: The degree of amplification was scored in four grades.−: No amplification was observed.

As shown in Table 1, the region of interest was efficiently amplifiedeven if a template having a high GC content was used. This amplificationwas accomplished by elevating the reaction temperature (from 55° C. to60° C.) and by using a primer having a higher Tm value as compared withan optimal primer for a reaction at 55° C. when the reaction was carriedat 60° C.

(3) The relationship between the length of an amplified fragment and theamount of the amplification product in the method for amplifying anucleotide sequence under high reaction temperature conditions wasexamined. First, a pair of primers having a sequence as shown in SEQ IDNO:70 or 71 of the Sequence Listing for amplifying a 800-bp region ofthe lambda DNA (Takara Shuzo) and a pair of primers having a sequence asshown in SEQ ID NO:72 or 73 of the Sequence Listing for amplifying a400-bp region of the lambda DNA were synthesized according to aconventional method. PCR was conducted using one of these primer pairsand the A DNA as a template to obtain an amplified fragment. Anamplified fragment of about 1.1 kbp was also prepared using thepUC19-911 plasmid as described in Example 5 (1) as a template and MF2(24) PCR primer and MR1 (24) PCR primer, which have sequences as shownin SEQ ID NOS:74 and 75 of the Sequence Listing, respectively. ThePCR-amplified fragments obtained by using the above-mentioned primerpairs have spacer sequences and the M13RV or M4 sequences on both ends.These fragments were used as templates for the present invention.

M13RV-2N 17mer primer or M13RV-2N 20mer primer as shown in (2) above wasused as a primer in this Example. In the primers, the first and secondbases from the 3′-terminus are ribonucleotides. A combination ofM13M4-3N 20mer primer having a sequence as shown in SEQ ID NO:76 of theSequence Listing and M13RV-3N 20mer primer having a sequence as shown inSEQ ID NO:77 of the Sequence Listing, and a combination of M13M4-3N24mer primer and M13RV-3N 24mer primer having sequences as shown in SEQID NO:78 and 79 of the Sequence Listing, respectively, were used foramplifying a region of about 1 kbp. In the primers, the first to thirdbases from the 3′-terminus are ribonucleotides. The reaction was carriedout as follows. 10 μl of a mixture of 10 pmol of the primer, about 20 ngof the template and 0.01% propylenediamine was denatured at 98° C. for 2minutes, and then cooled to 55° C. or 60° C. Thereafter, 34 mM Tricinebuffer (pH 8.7), 10 mM potassium chloride, 10 mM ammonium sulfate, 0.01%BSA, 1% DMSO, 4 mM magnesium acetate, 0.5 mM each of dNTPs, 11 U of BcaBEST DNA polymerase and 30 U of RNase H were added thereto to make thefinal reaction volume to 50 μl. The reaction mixture was incubated at55° C. or 60° C. for 1 hour. After the completion of the reaction, themixture was cooled to 4° C., and then 5 μl of a 0.5 M EDTA solution wasadded thereto to terminate the reaction. 3 μl of the reaction mixturewas subjected to electrophoresis on 3% NuSieve 3:1 agarose (TakaraShuzo) gel. The results are shown in Tables 2 and 3 below. TABLE 2Length of amplified fragment Reaction Primers and results temperatureused 400 bp 800 bp 55° C. M13RV-2N 17mer ++ ++ M13RV-2N 20mer ++ ++ 60°C. M13RV-2N 17mer + + M13RV-2N 20mer ++++ +++++ to ++++: The degree of amplification was scored in four grades.−: No amplification was observed.

As shown in Table 2, fragments for regions of 400 bp and 800 bp wereefficiently amplified by making the length of the primer foramplification from 17mer to 20mer and by elevating the reactiontemperature from 55° C. to 60° C. TABLE 3 Length of amplified ReactionPrimers fragment and results temperature used 1034 bp 55° C. M13RV-3N20mer & ++ M13M4-3N 20mer M13RV-3N 24mer & ++ M13M4-3N 24mer 65° C.M13RV-3N 20mer & + M13M4-3N 20mer M13RV-3N 24mer & ++++ M13M4-3N 24mer+ to ++++: The degree of amplification was scored in four grades.−: No amplification was observed.

Furthermore, as shown in Table 3, a fragment for a region of about 1 kbpwas efficiently amplified by making the length of the primer foramplification from 20mer to 24mer and by elevating the reactiontemperature from 55° C. to 65° C. Additionally, similar results wereobtained for amplification of a long-chain DNA fragment as described inExample 10 by using longer primers and an elevated reaction temperature.Increase in amplification efficiency was observed when a region of about2 kbp or longer was amplified.

Example 13

(1) Use of a heat-resistant DNA polymerase other than Bca BEST DNApolymerase in the method of the present invention was examined. Bst DNApolymerase (New England Biolabs) was used as a heat-resistant DNApolymerase. A pair of primers, 5′-ID primer and 3′-ID primer havingsequences as shown in SEQ ID NOS:80 and 81 of the Sequence Listing,respectively, were synthesized according to a conventional method. PCRwas carried out using the primer pair and a commercially available DNAfragment for cyclin A gene (Research Genetics) as a template, resultingin an amplified fragment of about 300 bp. The PCR-amplified fragmentobtained by using the primer pair has the M13RV sequences on both ends.The fragment was used as a template for the present invention.

M13RV-2N 17mer primer having a sequence as shown in SEQ ID NO:64 of theSequence Listing was used as a primer in this Example. In the primer,the first and second bases from the 3′-terminus are ribonucleotides. Thereaction was carried out as follows. 10 μl of a mixture of 20 μM of theprimer, about 20 ng of the template and 0.01% propylenediamine wasdenatured at 98° C. for 2 minutes, and then cooled to 55° C. Thereafter,34 mM Tricine buffer (pH 8.7), 10 mM potassium chloride, 10 mM ammoniumsulfate, 0.01% BSA, 1% DMSO, 4 mM magnesium acetate, 0.5 mM each ofdNTPs, 4, 8, 12 or 16 U of Bst DNA polymerase and 30 U of RNase H wereadded thereto to make the final reaction volume to 50 μl. As a control,a reaction mixture having a composition identical with theabove-mentioned one was prepared, except that 11 U of Bca BEST DNApolymerase was used. The reaction mixture was incubated at 55° C. for 1hour. After the completion of the reaction, the mixture was cooled to 4°C., and then 5 μl of a 0.5 M EDTA solution was added thereto toterminate the reaction. 3 μl of the reaction mixture was subjected toelectrophoresis on 3% NuSieve 3:1 agarose (Takara Shuzo) gel. As aresult, an amplified fragment of interest was obtained using each of thevarious units of Bst DNA polymerase. Thus, it was confirmed thatheat-resistant DNA polymerases can be preferably used in the method ofthe present invention.

(2) Use of a mesophilic DNA polymerase in the method of the presentinvention was examined. 5′3′exo activity (−) Klenow fragment (TakaraShuzo) was used as a mesophilic DNA polymerase. The DNA prepared in (1)above was used a template DNA for the method of the present invention.

M13RV-2N 16mer primer having a sequence as shown in SEQ ID NO:82 of theSequence Listing was used as a primer in this Example. In the primer,the first and second bases from the 3′-terminus are ribonucleotides. Thereaction was carried out as follows. 10 μl of a mixture of 20 μM of theprimer, about 20 ng of the template and 0.01% propylenediamine wasdenatured at 98° C. for 2 minutes, and then cooled to 40° C. Thereafter,34 mM Tricine buffer (pH 8.7), 10 mM potassium chloride, 10 mM ammoniumsulfate, 0.01% BSA, 1% DMSO, 4 mM magnesium acetate, 0.5 mM each ofdNTPs, 0, 2, 4, 6 or 8 U of Klenow fragment and 30 U of RNase H wereadded thereto to make the final reaction volume to 50 μl. The reactionmixture was incubated at 40° C. for 1 hour. After the completion of thereaction, the mixture was cooled to 4° C., and then 5 μl of a 0.5 M EDTAsolution was added thereto to terminate the reaction. 3 μl of thereaction mixture was subjected to electrophoresis on 3% NuSieve 3:1agarose (Takara Shuzo) gel. As a result, an amplified fragment ofinterest was obtained in cases where the various units of Klenowfragment were used excluding the case where no Klenow fragment wasadded. Thus, it was confirmed that mesophilic DNA polymerases can bepreferably used in the method of the present invention.

Example 14

Chimeric oligonucleotide primers to be used in the method of the presentinvention were examined. A DNA as a template and primers weresynthesized as described in Example 1 (1). The structures of the primersused in this Examples are described below in detail:

-   -   Primer Pair 1: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:2 or 3 of the Sequence Listing        and wholly composed of deoxyribonucleotides;    -   Primer Pair 2: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:83 or 84 of the Sequence Listing        in which the sixth and seventh deoxyribonucleotides from the        3′-terminus are ribonucleotides;    -   Primer Pair 3: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:85 or 86 of the Sequence Listing        in which the fifth and sixth deoxyribonucleotides from the        3′-terminus are ribonucleotides;    -   Primer Pair 4: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:87 or 88 of the Sequence Listing        in which the fourth and fifth deoxyribonucleotides from the        3′-terminus are ribonucleotides;    -   Primer Pair 5: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:89 or 90 of the Sequence Listing        in which the third and fourth deoxyribonucleotides from the        3′-terminus are ribonucleotides;    -   Primer Pair 6: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO: 91 or 92 of the Sequence Listing        in which the second and third deoxyribonucleotides from the        3′-terminus are ribonucleotides;    -   Primer Pair 7: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:93 or 94 of the Sequence Listing        in which the first and second deoxyribonucleotides from the        3′-terminus are ribonucleotides; and    -   Primer Pair 8: A combination of primers having a nucleotide        sequence as shown in SEQ ID NO:91 or 92 of the Sequence Listing        in which the second and third deoxyribonucleotides from the        3′-terminus are ribonucleotides and the phosphate bond on the        5′-terminal side of the third ribonucleotide from the        3′-terminus is replaced by a phosphorothioate bond.

Amplification conditions and detection method were as described inExample 1 (2) and (3). As a result, an amplified fragment having alength of interest was observed for each of the Primer Pairs 2-8. Forthe Primer Pairs 2-7, the amount of the amplification product increasedas the number of deoxyribonucleotides at the 3′-terminus decreased.Particularly, the most abundant amplification product was observed forthe Primer Pair 7 having no deoxyribonucleotide at the 3′-terminus. Onthe other hand, no amplified fragment was observed for the PrimerPair 1. Furthermore, the fact that amplified fragments of interest wereobserved for both of the Primer Pairs 6 and 8 confirms that both of amodified ribonucleotide and a unmodified ribonucleotide can bepreferably used as a ribonucleotide contained in a primer in the methodof the present invention.

Example 15

(1) The effect of the type of the buffer used in the method of thepresent invention was examined. Primers for amplifying λ DNA havingsequences represented by SEQ ID NOS:128 and 129 were used in thisExample. The reaction was carried out as follows. Briefly, 10 μl of amixture containing 120 pmol each of the primers, 0.01% aqueous solutionof propylenediamine and 10 ng or 1 ng of a DNA as a template washeat-denatured at 98 C for 2 minutes, and then cooled on ice to annealthe primers to the template DNA. An amplified product (1005 bp) obtainedby a PCR using λ DNA (Takara Shuzo) as a template and primersrepresented by SEQ ID NOS:130 and 131, which was then purified usingSuprec02 (Takara Shuzo), was used as the template.

After annealing, 40 μl of one of three types of buffers for reaction(42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 42.5 mMBicine-potassium hydroxide buffer (pH 8.3) and 42.5 mM HEPES-potassiumhydroxide buffer (pH 7.8)) each containing 0.625 mM each of dNTPs, 5.0mM magnesium acetate, 0.0125% bovine serum albumin (BSA), 1.25% dimethylsulfoxide (DMSO), 30 U of E. coli RNase H and 11 U of BcaBEST DNApolymerase was added to the mixture to make the final volume to 50 μl.The reaction mixtures were incubated at 60 C for 1 hour. After reaction,3 μl each of the reaction mixtures was subjected to electrophoresis on3.0% agarose gel for confirmation. Then, the amplified fragments ofinterest were observed for both of the amounts of the template. Inparticular, a greater amount of amplification product was obtained forthe reaction system containing Bicine-potassium hydroxide buffer (pH8.5).

(2) Improvement of reactivity by the use of HEPES-potassium hydroxidebuffer was examined. PUC19 plasmid DNA with a DNA fragment of about 150bp being inserted into the multi-cloning site was used as a template.This template was prepared as follows.

A PCR was carried out using pUC19 upper 150 PCR primer and pUC19 lowerPCR primer having sequences represented by SEQ ID NOS:223 and 224, and100 μg of pUC19 plasmid DNA as a template. The resulting amplifiedfragment was purified using Microcon-100, blunt-ended using DNA bluntingkit (Takara Shuzo) and subcloned into a HincII site of the plasmidpUC19. The plasmid with the amplified fragment being inserted was usedto transform Escherichia coli JM109. The transformant was cultured. Theplasmid having the inserted DNA was purified from the cells using QIAGENplasmid mini kit (Qiagen) and used as a template.

A DNA fragment PCR-amplified using the pUC19-150 plasmid DNA prepared asdescribed above as a template, and primers MCS-F and MCS-R havingnucleotide sequences represented by SEQ ID NOS:124 and 125 was used as atemplate. Primers MF2N3(24) and MR1N3(24) having nucleotide sequencesrepresented by SEQ ID NOS:126 and 127 were used as chimericoligonucleotide primers. The expected size of the amplified fragmentobtained using the combination of these primers was about 350 bp.

An HEPES-potassium hydoxide buffer system was selected as a buffer to beexamined. A potassium phosphate buffer system and a Tricine buffersystem were used as controls. The compositions of the reaction mixturesare shown below.

Reaction mixture A: 10 ng of the PCR-amplified fragment, 50 pmol each ofthe primers MF2N3(24) and MR1N3(24), 0.01% aqueous solution ofpropylenediamine and sterile distilled water to a reaction volume of 10μl.

Reaction mixtures B: The following three types were prepared.

Potassium phosphate buffer system: 40 μl of a reaction mixturecontaining, at final concentrations, 35 mM potassium phosphate buffer(pH 7.5), 1.25% DMSO, 0.0125% BSA, 5 mM magnesium acetate, 0.625 mM eachof dNTPs, 60 U of E. coli RNase H and 5.5 U of BcaBEST DNA polymerasewas prepared.

Tricine buffer system: 40 μl of a reaction mixture containing, at finalconcentrations, 42.5 mM Tricine buffer (pH 8.7), 12.5 mM potassiumchloride, 12.5 mM ammonium sulfate, 1.25% DMSO, 0.0125% BSA, 5 mMmagnesium acetate, 0.625 mM each of dNTPs, 30 U of E. coli RNase H and5.5 U of BcaBEST DNA polymerase was prepared.

HEPES-potassium hydroxide buffer system: 40 μl of a reaction mixturecontaining, at final concentrations, 25 mM HEPES-potassium hydroxidebuffer (pH 7.8), 125 mM potassium acetate, 1.25% DMSO, 0.0125% BSA, 5 mMmagnesium acetate, 0.625 mM each of dNTPs, 30 U of E. coli RNase H and5.5 U of BcaBEST DNA polymerase was prepared.

The reaction mixture A was heat-denatured at 98 C for 2 minutes, cooledto 60 C or 65 C and then allowed to stand on ice. One of the reactionmixtures B was added to the reaction mixture A on ice and mixed to makethe reaction volume to 50 μl. The reaction mixtures were incubated at 60C or 65 C for 1 hour. After reaction, they were cooled to 4 C, and 1/10volume of 0.5 M EDTA was added to each of the mixtures to terminate thereaction. 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3% NuSieve 3:1 agarose gel. As a result, theamplified fragments of interest were observed for the three buffersystems regardless of the reaction temperature used. In particular, thelargest amount of the amplification product and the highest reactivitywere observed for the HEPES-potassium hydroxide buffer system in thisExample.

Example 16

(1) Conditions used in the method of the present invention for annealingprimers to a template were examined. Primers having nucleotide sequencesrepresented by SEQ ID NOS:132 and 133 based on the partial nucleotidesequence of a bacterium Flavobacterium sp. SA-0082 as described in WO97/32010 (deposited at the International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology, AISTTsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken305-8566, Japan on Mar. 29, 1995 under accession number FERM P-14872,and deposited at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology under accessionnumber FERM BP-5402 (date of request for transmission to internationaldepositary authority: Feb. 15, 1996)) were used. An amplified product(573 bp) obtained by a PCR using a genomic DNA from Flavobacterium sp.SA-0082 as a template and a combination of primers represented by SEQ IDNOS:134 and 135, which was then purified using Suprec02 (Takara Shuzo),was used as the DNA as the template in this Example. The reaction wascarried out as follows. Briefly, 2 μl of one of two annealing solutions(500 mM potassium chloride and 8 μM spermidine, or 0.05%propylenediamine) was added to 120 pmol each of the primers. A finalvolume of 10 μl of each of the mixtures further containing 10 ng or 1 ngof the PCR-amplified fragment was heat-denatured at 98 C for 2 minute.After denaturation, the mixtures were rapidly cooled on ice to annealthe primers to the template.

After annealing, 40 μl of one of three types of buffers (42.5 mMTricine-potassium hydroxide buffer (pH 8.5), 42.5 mM Bicine-potassiumhydroxide buffer (pH 8.3) and 42.5 mM HEPES-potassium hydroxide buffer(pH 7.8)) each containing 0.625 mM each of dNTPs, 5.0 mM magnesiumacetate, 0.0125% bovine serum albumin (BSA), 1.25% dimethyl sulfoxide(DMSO), 30 U of E. coli RNase H and 11 U of BcaBEST DNA polymerase wasadded to the mixture to make the final volume to 50 μl. The reactionmixtures were incubated at 52 C for 1 hour. After reaction, 3 μl each ofthe reaction mixtures was subjected to electrophoresis on 3.0% agarosegel. The results are shown in FIG. 1. FIG. 1 is a photograph ofelectrophoresis of reactions using the respective combinations of theannealing solutions and the buffers. Lane 1: molecular weight marker(100 bp ladder, Takara Shuzo); lane 2: propylenediamine/Tricine(template: 10 ng); lane 3: propylenediamine/HEPES (template: 10 ng);lane 4: propylenediamine/HEPES (template: 1 ng); lane 5:propylenediamine/Bicine (template: 10 ng); lane 6:propylenediamine/Bicine (template: 1 ng); lane 7: 500 mM potassiumchloride and 8 μM spermidine/Bicine (template: 10 ng); lane 8: 500 mMpotassium chloride and 8 μM spermidine/Bicine (template: 1 ng); lane 9:molecular weight marker (100 bp ladder); lane 10:propylenediamine/Tricine (template: 1 ng); lane 11: 500 mM potassiumchloride and 8 μM spermidine/Tricine (template: 1 ng); lane 12:propylenediamine/HEPES (template 1 ng); lane 13: 500 mM potassiumchloride and 8 μM spermidine/HEPES (template: 1 ng); lane 14:propylenediamine/Bicine (template: 1 ng); and lane 15: 500 mM potassiumchloride and 8 μM spermidine/Bicine (template: 1 ng).

As shown in FIG. 1, a greater amount of the amplification product ofinterest was obtained when the annealing solution containing 500 mMpotassium chloride+8 μM spermidine was used for the annealing of theprimers to the template DNA for either of the three buffers regardlessof the amount of the DNA as the template. In particular, the combinationof the annealing solution containing 500 mM potassium chloride+8 μMspermidine and the Bicine-potassium hydroxide buffer yielded goodresults in this

EXAMPLE

(2) The effect of an annealing solution in case where a PCR-amplifiedfragment from λ DNA was used as a template was examined. The chimericoligonucleotide primers as described in Example 15(1) were used in thisExample. The PCR-amplified fragment as prepared in Example 15(1) or λDNA was used as a template DNA. The reaction was carried out as follows.Briefly, 2 μl of one of three types of annealing solutions (500 mMpotassium chloride and 8 μM spermidine, 0.05% propylenediamine, orsterile water) was added to 120 pmol each of the primers. 10 μl of themixtures further containing 10 ng or 1 ng of the PCR-amplified fragmentwere prepared. The mixtures were heat-denatured at 98 C for 2 minutes,and then rapidly cooled on ice to anneal the primers to the template.

After annealing, 40 μl of one of three types of buffers (42.5 mMTricine-potassium hydroxide buffer (pH 8.5), 42.5 mM Bicine-potassiumhydroxide buffer (pH 8.3) and 42.5 mM HEPES-potassium hydroxide buffer(pH 7.8)) each containing 0.625 mM each of dNTPs, 5.0 mM magnesiumacetate, 0.0125% BSA, 1.25% DMSO, 30 U of E. coli RNase H and 11 U ofBcaBEST DNA polymerase was added to the mixture to make the final volumeto 50 μl. The reaction mixtures were incubated at 60 C for 1 hour. Afterreaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. The results are shown in FIG. 2.FIG. 2 is a photograph of electrophoresis showing the results for theexamination of combinations of the amounts of the template, the reactionbuffers and the annealing solutions. Lane 1: marker (100 bp ladder);lane 2: 10 ng of the template, a combination of Tricine/500 mM potassiumchloride and 8 μM spermidine; lane 3: 1 ng of the template, acombination of Tricine/500 mM potassium chloride and 8 μM spermidine;lane 4: 10 ng of the template, a combination of Bicine/500 mM potassiumchloride and 8 μM spermidine; lane 5: 1 ng of the template, acombination of Bicine/500 mM potassium chloride and 8 μM spermidine;lane 6: 10 ng of the template, a combination of HEPES/500 mM potassiumchloride and 8 μM spermidine; lane 7: 1 ng of the template, acombination of HEPES/500 mM potassium chloride and 8 μM spermidine; lane8: molecular weight marker (100 bp ladder); lane 9: 10 ng of thetemplate, a combination of Tricine/propylenediamine; lane 10: 1 ng ofthe template, a combination of Tricine/propylenediamine; lane 11: 10 ngof the template, a combination of Bicine/propylenediamine; lane 12: 1 ngof the template, a combination of Bicine/propylenediamine; lane 13: 10ng of the template, a combination of HEPES/propylenediamine; lane 14: 1ng of the template, a combination of HEPES/propylenediamine; lane 15:molecular weight marker (100 bp ladder); lane 16: 10 ng of the template,a combination of Tricine/water; lane 17: 1 ng of the template, acombination of Tricine/water; lane 18: 10 ng of the template, acombination of Bicine/water; lane 19: 1 ng of the template, acombination of Bicine/water; lane 20: 10 ng of the template, acombination of HEPES/water; and lane 21: 1 ng of the template, acombination of HEPES/water.

As shown in FIG. 2, the amplified fragments of interest were observedfor the respective combinations of the three buffers and the threeannealing solutions regardless of the amount of the template DNA. Inparticular, it was confirmed that a greater amount of the amplifiedfragment was obtained using the combination of the Bicine buffer and theannealing solution containing 500 mM potassium chloride and 8 μMspermidine.

Example 17

The effect of the presence of an inhibitor of reverse transcriptase(RTase) on the method of the present invention was examined.Phosphonoformic acid (PFA) was used as an inhibitor of RTase. Primersrepresented by SEQ ID NOS:136 and 137 were used in this Example. Anamplified product (576 bp) obtained by a PCR using a genomic DNA fromenterohemorrhagic Escherichia coli O-157 as a template and primersrepresented by SEQ ID NOS:138 and 139, which was then purified usingSuprec02 (Takara Shuzo), was used as the template DNA. The reaction wascarried out as follows. Briefly, 10 μl of a mixture prepared by adding 1ng of the PCR-amplified fragment to 120 pmol each of the primers and 2μl of an annealing solutions containing 500 mM potassium chloride and 8μM spermidine was heat-denatured at 98 C for 2 minutes, and then rapidlycooled on ice to anneal the primers to the template.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% bovine serum albumin (BSA), 1.25% dimethyl sulfoxide(DMSO), 30 U of E. coli RNase H and 11 U of BcaBEST DNA polymerase aswell as PFA to a concentration of 500 μg/ml or 50 μg/ml were added tothe annealing mixture to make the final volume to 50 μl. The reactionmixtures were incubated at 55 C for 1 hour. A system without theaddition of PFA was also prepared as a control. After reaction, 9 μleach of the reaction mixtures was subjected to electrophoresis on 3.0%agarose gel. The results are shown in FIG. 3. FIG. 3 is a photograph ofelectrophoresis which shows the effect of the inhibitor of RTase. Lane1: molecular weight marker (100 bp ladder); lane 2: without the additionof PFA; lane 3: with the addition of PFA at a concentration of 500μg/ml; lane 4: with the addition of PFA at a concentration of 50 μg/ml.

As shown in FIG. 3, when PFA was added, non-specific amplification wassuppressed and the amplified fragment of interest was observed. Inparticular, it was confirmed that the non-specific amplificationproducts which were observed in the system without the addition of PFAwas not observed and the amplified fragment of interest was clearlyamplified in the system to which PFA was added at a concentration of 500μg/ml.

Example 18

The relationship between the length of a fragment to be amplified andthe detection sensitivity in the method of the present invention wasexamined.

(1) Primers for amplifying Escherichia coli O-157 vero toxin representedby SEQ ID NOS:140-142 were synthesized. The chimeric oligonucleotideprimers used in Example 17 were also used. The length of a fragment tobe amplified using each of the combinations of primers was as follows:247 bp (SEQ ID NOS:140 and 137); 168 bp (SEQ ID NOS:141 and 142); 206 bp(SEQ ID NOS:141 and 137); 135 bp (SEQ ID NOS:136 and 142); and 173 bp(SEQ ID NOS:136 and 137). The purified PCR-amplified 576-bp fragmentprepared in Example 17 was used as a template DNA in this Example. Thereaction was carried out as follows. Briefly, 10 μl of a mixturecontaining 60 pmol each of the primers, 2 μl of 0.05% aqueous solutionof propylenediamine and 10 fg to 10 ng of the PCR-amplified fragment washeat-denatured at 98 C for 2 minutes and then cooled to 55 C in ThermalCycler Personal (Takara Shuzo) to anneal the primers to the template.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% bovine serum albumin (BSA), 1.25% dimethyl sulfoxide(DMSO), 30 U of E. coli RNase H, 5.5 U of BcaBEST DNA polymerase andsterile water was added to the mixture to make the final volume to 50μl. The reaction mixtures were incubated at 55 C for 1 hour. Afterreaction, 5 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. As a control, detection of 10 fg to1 pg of the PCR-amplified fragment was carried out using primersrepresented by SEQ ID NOS:143 and 144. A 135-bp fragment is amplifiedusing the combination of these primers. 50 μl of a PCR solutioncontaining 60 pmol each of the primers, 5 μl of 10×Ex Taq buffer (TakaraShuzo), 1.25 U of TaKaRa Ex Taq DNA polymerase (Takara Shuzo) and 0.2 mMeach of dNTPs was prepared. The PCR was carried out as follows: 25 or 30cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 C for 30seconds (2 min. 38 sec./cycle). After reaction, 1 μl (ICAN) or 5 μl(PCR) each of the reaction mixtures was subjected to electrophoresis on3.0% agarose gel. The results are shown in FIG. 4 and Table 4. TABLE 4Amplification size (bp) Detection limit ICAN (total time: 70 minutes)247 100 pg 168 100 fg 206 100 pg 135 10 fg 173 100 fg PCR (25 cycles;total time: about 66 minutes) 135 100 fg PCR (30 cycles; total time:about 80 minutes) 135 10 fg

FIG. 4 is a photograph of electrophoresis which shows the detectionlimits for amplifying a chain length of 135 bp accomplished by the ICAN( 1/50 of the reaction mixture was loaded) and the PCR ( 1/10 of thereaction mixture was loaded). Lane 1: molecular weight marker (100 bpladder); lane 2: ICAN using 1 pg of the template; lane 3: ICAN using 100fg of the template; lane 4: ICAN using 10 fg of the template; lane 5:PCR (25 cycles) using 1 pg of the template; lane 6: PCR (25 cycles)using 100 fg of the template; lane 7: PCR (25 cycles) using 10 fg of thetemplate; lane 8: PCR (30 cycles) using 1 pg of the template; lane 9:PCR (30 cycles) using 100 fg of the template; and lane 10: PCR (30cycles) using 10 fg of the template.

As shown in Table 4, it was confirmed that almost the same detectionsensitivity as that of the PCR was accomplished by the ICAN.Furthermore, the total reaction time for the PCR was about 80 minutes,whereas the reaction time required for accomplishing the same detectionsensitivity using the method of the present invention was 70 minutes,confirming that the reaction time can be shortened by using the methodof the present invention.

(2) Primers for amplifying λ DNA having nucleotide sequences representedby SEQ ID NOS:128, 129 and 145 were synthesized. The length of afragment to be amplified using each of the combinations of primers wasas follows: 151 bp (SEQ ID NOS:128 and 129); and 125 bp (SEQ ID NOS:145and 129). The template DNA prepared in Example 15(1) was used in thisExample. The reaction was carried out as follows. Briefly, 2 μl of anannealing solution (500 mM potassium chloride and 8 μM spermidine), 1 fgto 1 ng of the template were added to 120 pmol each of the primers andfill up to 10 μl by sterile water. The mixture was heat-denatured at 98C for 2 minutes, and then rapidly cooled on ice to anneal the primers tothe template.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% BSA, 1.25% DMSO, 30 U of E. coli RNase H and 11 U ofBcaBEST DNA polymerase was added to the mixture to make the final volumeto 50 μl. The reaction mixtures were incubated at 60 C for 1 hour. Afterreaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. The results are shown in Table 5.TABLE 5 Amplification size (bp) Detection limit 125 10 fg 151 100 fg

As shown in Table 5, it was confirmed that, when λ DNA was used as atemplate, a detection sensitivity of as low as 10 fg could be alsoaccomplished by examining the optimal region.

(3) A plasmid was prepared by inserting an amplified fragment (length:340 bp) into a plasmid T7Blue T-Vector (Takara Shuzo). The fragment wasprepared as described in JP-A 9-140383 using synthetic primers foramplifying a chrysanthemum viroid gene having nucleotide sequencesrepresented by SEQ ID NOS:146 and 147, and an RNA from chrysanthemuminfected with the viroid as a template. The plasmid was used totransform Escherichia coli JM109 competent cells (Takara Shuzo). Thetransformant was cultured in 5 ml of LB medium at 37 C for 16 hour. Theplasmid was purified from the collected cells using QIAGEN Plasmid MiniKit (Qiagen) according to the manual. Dilutions containing 0 fg, 1 fg,10 fg, 100 fg, 1 pg, 10 pg, 100 pg or 1 ng of the plasmid in 1 μl ofsterile water were prepared based on the concentration of the plasmid asmeasured using Beckman DU-600 (Beckman). 1 μl of one of the thusprepared plasmid solutions was used as a template for 50 μl each of ICANreaction systems. Primers CSVD-F2 and CSVD-R6 having nucleotidesequences represented by SEQ ID NOS:148 and 149 were used in thisExample. The reaction was carried out as follows. Briefly, 10 μl of amixture containing 50 pmol each of the primers, 1 μl of the preparedplasmid solution and propylenediamine at a final concentration of 0.01%was prepared. The mixture was heated at 98 C for 2 minutes, cooled to 60C, incubated at the temperature for 1 minute in Thermal Cycler Personal(Takara Shuzo), and then placed on ice.

After annealing, at final concentrations, 20 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 1% DMSO, 0.01% BSA,4 mM magnesium acetate, 500 μM each of dNTPs, 30 U of E. coli RNase Hand 5.5 U of BcaBEST DNA polymerase were added to the mixture to makethe final volume to 50 μl with sterile water. The reaction mixtures wereplaced in Thermal Cycler Personal which had been set at 60 C and reactedfor 60 minutes. After reaction, 3 μl each of the reaction mixtures wassubjected to electrophoresis on 3% NuSieve 3:1 agarose. As a result, theamplification products of interest (about 90 bp, about 70 bp and about50 bp) were observed using a template at a concentration of 10 fg.

Example 19

Primers to be used in the method of the present invention were examined.

(1) The Tm value of a primer and the reaction temperature were examined.Primers for amplifying Flavobacterium sp. SA-0082 having nucleotidesequences represented by SEQ ID NOS:132 and 150-152 were synthesized.These primers were constructed such that a region of 160 bp or shorterhaving a GC content of about 20% was amplified. The length of a fragmentto be amplified using each of the combinations of primers was asfollows: 126 bp (SEQ ID NOS:132 and 151); 158 bp (SEQ ID NOS:132 and152); 91 bp (SEQ ID NOS:150 and 151); and 123 bp (SEQ ID NOS:150 and152). The PCR-amplification product as prepared in Example 16(1) wasused as a DNA as the template in this Example. The reaction was carriedout as follows. Briefly, 10 μl of a mixture containing 120 pmol each ofthe primers, 2 μl of one of three types of annealing solutions (500 mMpotassium chloride and 8 μM spermidine, 0.05% propylenediamine, orwater) and 1 fg to 10 ng of the template was prepared. The mixture washeat-denatured at 98 C for 2 minutes, and then cooled on ice to annealthe primers to the template.

After annealing, 40 μl of one of three types of buffers (17 mMTricine-potassium hydroxide buffer (pH 8.5), 17 mM Bicine-potassiumhydroxide buffer (pH 8.3) and 20 mM HEPES-potassium hydroxide buffer (pH7.8)) containing 0.625 mM each of dNTPs, 5.0 mM magnesium acetate,0.0125% bovine serum albumin (BSA), 1.25% dimethyl sulfoxide (DMSO), 30U of E. coli RNase H and 11 U of BcaBEST DNA polymerase was added to themixture to make the final volume to 50 μl. The reaction mixtures wereincubated at 52, 55 or 60 C for 1 hour. After reaction, 3 μl each of thereaction mixtures was subjected to electrophoresis on 3.0% agarose gel.As a result, the amplified fragment of interest was observed using thereaction temperature of 52 C. In particular, a greater amount ofamplified fragment of interest was obtained using the combination of theannealing solution containing 500 mM potassium chloride and 8 μMspermidine and Tricine or Bicine buffer. The primer pair, the length ofthe amplified fragment and the detection sensitivity for the reactiontemperature of 52 C are shown in FIG. 5 and Table 6. TABLE 6Amplification size (bp) Detection limit 126 100 fg 158 1 pg 91 1 fg 123100 fg

FIG. 5 is a photograph of electrophoresis which shows the relationshipbetween the length of the amplified fragment and the amount of the DNAas the template when an AT-rich region was amplified. Lane 1: molecularweight marker (100 bp ladder); lane 2: amplification of a fragment of 91bp in length using 1 pg of the template; lane 3: amplification of afragment of 91 bp in length using 100 fg of the template; lane 4:amplification of a fragment of 91 bp in length using 10 fg of thetemplate; lane 5: amplification of a fragment of 91 bp in length using 1fg of the template; lane 6: amplification of a fragment of 123 bp inlength using 1 pg of the template; lane 7: amplification of a fragmentof 123 bp in length using 100 fg of the template; lane 8: amplificationof a fragment of 123 bp in length using 10 fg of the template; lane 9:amplification of a fragment of 126 bp in length using 1 pg of thetemplate; lane 10: amplification of a fragment of 126 bp in length using100 fg of the template; lane 11: amplification of a fragment of 126 bpin length using 10 fg of the template; lane 12: amplification of afragment of 158 bp in length using 1 pg of the template; lane 13:amplification of a fragment of 158 bp in length using 100 fg of thetemplate; and lane 14: amplification of a fragment of 158 bp in lengthusing 10 fg of the template.

As shown in FIG. 5 and Table 6, it was demonstrated that good resultswere yielded by lower the reaction temperature depending on the Tm valueof the primer when the method of the present invention was carried outusing an AT-rich template and a set of AT-rich primers.

(2) The higher-order structure of the primer may influence thereactivity in the method of the present invention. Then, modification ofthe primer for avoiding the formation of the higher-order structure ofthe primer and for making the primer readily anneal to the objectivetemplate was examined. Primers represented by SEQ ID NOS:136, 137 and153-158 were used. Specifically, a primer having a nucleotide sequencerepresented by SEQ ID NOS:136, primers 120I4, 121I5 and 122I6 which areprimers having nucleotide sequences represented by SEQ ID NOS:153 to 155and having an inosine deoxynucleotide at the fourth, fifth or sixth basefrom the 3′-terminus, a primer having a nucleotide sequence representedby SEQ ID NO:193, and primers 123I4, 124I5 and 125I6 which are primershaving nucleotide sequences represented by SEQ ID NOS:156 to 158 andhaving an inosine deoxynucleotide at the fourth, fifth or sixth basefrom the 3′-terminus were used. The DNA as the template as prepared inExample 17 was used in this Example. The reaction was carried out asfollows. Briefly, 10 μl of a mixture containing 50 pmol each of theprimers, 2 μl of 0.05% aqueous solution of propylenediamine, 1 ng to 10ng of the DNA as the template and sterile distilled water was heated at98 C for 2 minutes, cooled to 55 C, and incubated at the temperature for1 minute using a thermal cycler (GeneAmp PCR System 9600, AppliedBiosystems).

After annealing, 0.625 mM each of dNTPs, 42.5 mM Tricine-potassiumhydroxide buffer (pH 8.5), 5.0 mM magnesium acetate, 0.0125% BSA, 1.25%DMSO, 30 U of E. coli RNase H or 5 U of a heat-resistant RNase H fromThermus thermophilus (Tth) (Toyobo, hereinafter referred to as Tth RNaseH) and 5.5 U of BcaBEST DNA polymerase were added to the mixture to makethe final volume to 50 μl with sterile water. The reaction mixtures wereincubated at 55 C for 1 hour. After reaction, 5 μl each of the reactionmixtures was subjected to electrophoresis on 3.0% agarose gel. Theresults are shown in FIG. 6.

FIG. 6 is a photograph of electrophoresis which shows the effects onchimeric oligonucleotide primers containing inosine deoxynucleotideswhen E. coli RNase H and Tth RNase H were used. Lanes 2 to 9 representthe results obtained using E. coli RNase H. Lanes 10 to 17 represent theresults obtained using Tth RNase H. Lane 1: molecular weight marker (100bp ladder); lane 2: a pair of primers represented by SEQ ID NOS:136 and137, 1 ng of the template; lane 3: a pair of primers 120I4 and 123I4, 1ng of the template; lane 4: a pair of primers 121I5 and 124I5, 1 ng ofthe template; lane 5: a pair of primers 122I6 and 125I6, 1 ng of thetemplate; lane 6: a pair of primers represented by SEQ ID NOS:136 and137, 10 ng of the template; lane 7: a pair of primers 120I4 and 123I4,10 ng of the template; lane 8: a pair of primers 121I5 and 124I5, 10 ngof the template; lane 9: a pair of primers 122I6 and 125I6, 10 ng of thetemplate; lane 10: a pair of primers represented by SEQ ID NOS:136 and137, 1 ng of the template; lane 11: a pair of primers 120I4 and 123I4, 1ng of the template; lane 12: a pair of primers 121I5 and 124I5, 1 ng ofthe template; lane 13: a pair of primers 122I6 and 125I6, 1 ng of thetemplate; lane 14: a pair of primers represented by SEQ ID NOS:136 and137, 10 ng of the template; lane 15: a pair of primers 120I4 and 123I4,10 ng of the template; lane 16: a pair of primers 121I5 and 124I5, 10 ngof the template; and lane 17: a pair of primers 122I6 and 125I6, 10 ngof the template.

As shown in FIG. 6, when primers having inosines being incorporated atfourth or fifth base from the 3′-termini of the primers were used, theamplification products of interest were observed using either of E. coliRNase H and the heat-resistant RNase H from Thermus thermophilusregardless of the amount of the template. These results demonstrate thatthe reactivity of the ICAN is improved by incorporating inosine at anappropriate position.

(3) Primers were examined for the same purpose as that in (2) above.Oligonucleotide primers 1S and 4S having nucleotide sequencesrepresented by SEQ ID NOS:173 and 174 in which the three bases at the3′-terminus were α-S (or alpha-thio) ribonucleotides, i.e., which had5′-phosphothioate bonds in the RNA moieties, were synthesized. Inaddition, oligonucleotide primers 1N3N3 and 4N3N3 having nucleotidesequences represented by SEQ ID NOS:159 and 160 and havingribonucleotides at the three bases from the 3′-terminus and in a portionof the sequence of the deoxyribonucleotide moiety, i.e., ribonucleotidesat eleventh to thirteenth bases from the 3′-terminus of the primer, weresynthesized. The template DNA as prepared in Example 17 was used. Thereaction was carried out as follows. Briefly, 10 μl of a mixturecontaining 50 pmol each of the primers, 2 μl of 0.05% aqueous solutionof propylenediamine, 10 ng of the DNA as the template and sterile waterwas heated at 98 C for 2 minutes using a thermal cycler, and then placedon ice for cooling.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% BSA, 1.25% DMSO, 30 U of E. coli RNase H or 5 U of TthRNase H and 5.5 U of BcaBEST DNA polymerase was added to the mixture tomake the final volume to 50 μl with sterile water. The reaction mixtureswere incubated at 55 C for 1 hour in a thermal cycler.

After reaction, 5 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. As a result, the amplificationproduct was clearly observed at the expected position using thecombination of primers 1S and 4S or 1N3N3 and 4N3N3 regardless of thetype of the RNase H used. These results confirmed that modification of aprimer at its 3′-terminus with 5′-phophothioate was effective for themethod of the present invention. Additionally, it was confirmed thatsubstitution with a ribonucleotide at an appropriate internal positionin addition to the 3′-terminus of a primer was effective for improvingthe reactivity of the method of the present invention.

Example 20

Use of a DNA polymerase having RNase H activity in the presence of aspecific metal ion in the method of the present invention was examined.10 μl of a mixture containing 120 pmol each of the chimericoligonucleotide primers used in Example 15(1), 2 μl of an annealingsolution containing 500 mM potassium chloride and 8 μM spermidine, 1 ngof the DNA as the template used in Example 15(1) and sterile water washeat-denatured at 98 C for 2 minutes, and then rapidly cooled on ice toanneal the primers to the template. After annealing, 40 μl of a mixturecontaining 0.625 mM each of dNTPs, 42.5 mM Tricine-potassium hydroxidebuffer (pH 8.5), 5.0 mM magnesium acetate, 0.0125% bovine serum albumin(BSA), 1.0% dimethyl sulfoxide (DMSO) and 11 U of BcaBEST DNApolymerase, and manganese chloride (Nacalai Tesque) at a finalconcentration of 0.5, 2.5, 5.0 or 10 mM were added to the mixture tomake the final volume to 50 μl with sterile water. The reaction mixtureswere incubated at 60 C for 1 hour. In addition, a mixture without theaddition of manganese chloride, and a mixture to which 30 U of E. coliRNase H was added but manganese chloride was not added were prepared ascontrols. After reaction, 3 μl each of the reaction mixtures wassubjected to electrophoresis on 3.0% agarose gel. The results are shownin FIG. 7.

FIG. 7 is a photograph of electrophoresis which shows the results of theICAN utilizing the RNase H activity of BcaBEST DNA polymerase. Lane 1:molecular weight marker (100 bp ladder); lane 2: without the addition ofmanganese chloride/with the addition of E. coli RNase H; lane 3: withoutthe addition of manganese chloride/without the addition of E. coli RNaseH; lane 4: with the addition of 0.5 mM manganese chloride/without theaddition of E. coli RNase H; lane 5: with the addition of 2.5 mMmanganese chloride/without the addition of E. coli RNase H; lane 6: withthe addition of 5.0 mM manganese chloride/without the addition of E.coli RNase H; and lane 7: with the addition of 10.0 mM manganesechloride/without the addition of E. coli RNase H.

As shown in FIG. 7, the amplification product of interest was observedfor a reaction system to which manganese chloride was added at aconcentration of 2.5 mM in the absence of E. coli RNase H.

Example 21

The method of the present invention was examined using a practicalbiological sample.

(1) Detection was carried out using a hot water-extract prepared from aculture of enterohemorrhagic Escherichia coli O-157 (ATCC accessionnumber 43895) as a template. Enterohemorrhagic Escherichia coli O-157was cultured in mEC medium containing novobiocin at 42 C for 18 hours,and then heated at 95 C for 10 minutes. Hot water-extracts of O-157corresponding to 0, 1, 10, 10², 10³, 10⁴ or 10⁵ cells were prepared bydiluting the extract with sterile water. Vero toxin 2 (VT2) gene wasamplified using one of these hot water-extracts of O-157 under the sameconditions as those in Example 18(1). In addition, a PCR was carried outusing the same template under the conditions as described in Example18(1) as a control. After reaction, 1 μl (ICAN) or 5 μl (PCR) each ofthe reaction mixtures was subjected to electrophoresis on 3.0% agarosegel. The results are shown in Table 7 and FIG. 8. TABLE 7 Amplificationsize (bp) Detection limit (cells) ICAN (total time: 70 minutes) 135 10²173 10³ PCR (25 cycles; total time: about 66 minutes) 135 10³ PCR (30cycles; total time: about 80 minutes) 135 10²

FIG. 8 is a photograph of electrophoresis which shows the detection ofEscherichia coli O157 using ICAN or PCR. The chain length to beamplified was 135 bp. Lane 1: molecular weight marker (100 bp ladder);lane 2: ICAN for 10⁴ cells; lane 3: ICAN for 10³ cells; lane 4: ICAN for10² cells; lane 5: PCR of 25 cycles for 10⁴ cells; lane 6: PCR of 25cycles for 10³ cells; lane 7: PCR of 25 cycles for 10² cells; lane 8:PCR of 30 cycles for 10⁴ cells; lane 9: PCR of 30 cycles for 10³ cells;and lane 10: PCR of 30 cycles for 10² cells.

As shown in Table 7 and FIG. 8, it was confirmed that the detectionsensitivity of the detection method of the present invention wasequivalent to that of the PCR, and that the method of the presentinvention required shorter time for detection as compared with thatrequired for the PCR.

(2) λ DNA was detected using the primers represented by SEQ ID NOS:129and 145 used in Examples 15 and 17. The reaction was carried out asfollows. Briefly, 10 μl of a mixture containing 120 pmol each of theprimers, 2 μl of an annealing solution containing 500 mM potassiumchloride and 8 μM spermidine, 10 fg to 1 ng of λ DNA (Takara Shuzo) andsterile water was prepared. The mixture was heat-denatured at 98 C for 2minutes, and then rapidly cooled on ice to anneal the primers to thetemplate.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% bovine serum albumin (BSA), 1.25% dimethyl sulfoxide(DMSO), 30 U of E. coli RNase H and 11 U of BcaBEST DNA polymerase wasadded to the mixture to make the final volume to 50 μl with sterilewater. The reaction mixtures were incubated at 60 C for 1 hour. Afterreaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel. The results are shown in Table 8.TABLE 8 Amplification size (bp) Detection limit 125 1 pg

As shown in Table 8, it was confirmed that the method of the presentinvention was effective in detecting λ DNA.

(3) Detection was carried out using a genomic DNA from a bacteriumFlavobacterium sp. SA-0082 as a template and the primers represented bySEQ ID NOS:150 and 151 used in Example 19(1). The genomic DNA as atemplate was prepared according to a conventional method from thebacterium of genus Flavobacterium cultured as described in WO 97/32010.The reaction was carried out as follows. Briefly, 10 μl of a mixturecontaining 120 pmol each of the primers, 2 μl of an annealing solutioncontaining 500 mM potassium chloride and 8 μM spermidine, 10 fg to 1 ngof the genomic DNA and sterile water was prepared. The mixture washeat-denatured at 98 C for 2 minutes, and then rapidly cooled on ice toanneal the primers to the template.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% BSA, 1.25% DMSO, 30 U of E. coli RNase H and 11 U ofBcaBEST DNA polymerase was added to the mixture to make the final volumeto 50 μl with sterile water. The reaction mixtures were incubated at 52C for 1 hour. After reaction, 3 μl each of the reaction mixtures wassubjected to electrophoresis on 3.0% agarose gel. The results are shownin Table 9 and FIG. 9. TABLE 9 Amplification size (bp) Detection limit91 100 fg

FIG. 9 is a photograph of electrophoresis which shows the detection of abacterium of genus Flavobacterium. Lane 1: molecular weight marker (100bp ladder); lane 2: 1 ng of the template; lane 3: 10 pg of the template;lane 4: 1 pg of the template; lane 5: 100 fg of the template; and lane6: 10 fg of the template.

As shown in Table 9 and FIG. 9, it was confirmed that the method of thepresent invention was effective in detecting a bacterium.

Example 22

A method for detecting a target nucleic acid in which the amplificationmethod of the present invention and a hybridization method were combinedwas examined. Enterohemorrhagic Escherichia coli O-157 was selected as atarget. The DNA as the template was prepared as described in Example21(1). A region of about 100 bp having a GC content of about 40% wasselected as a fragment to be amplified. Primers VT2-IF20 and VT2-IR20-2having nucleotide sequences represented by SEQ ID NOS:140 and 161 wereused as primers. The reaction was carried out as follows. Briefly, 10 μlof a mixture containing 50 pmol each of the primers VT2-IF20 andVT2-IR20-2, an annealing solution containing propylenediamine at a finalconcentration of 0.01%, one of the hot water-extracts corresponding to 0to 10⁴ cells and sterile water was prepared. The mixture washeat-denatured at 98 C for 2 minutes, cooled to 55 C and incubated atthe temperature for 1 minute in Thermal Cycler Personal (Takara Shuzo),and then placed on ice for annealing.

After annealing, at final concentrations, 20 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 1% dimethylsulfoxide (DMSO), 0.01% bovine serum albumin (BSA), 4 mM magnesiumacetate, 500 μM each of dNTPs, 30 U of E. coli RNase H and 5.5 U ofBcaBEST DNA polymerase was added to the mixture to make the final volumeto 50 μl with sterile water. The reaction mixtures were placed onThermal Cycler Personal which had been set at 55 C and incubated at thetemperature for 60 minutes. As a control, a PCR was carried out usingO-157 Typing Set (Takara Shuzo) according to the manual using ThermalCycler Personal. The PCR was carried out as follows: 35 cycles of 94 Cfor 1 minute, 55 C for 1 minute and 72 C for 1 minute. This reactionrequired about 4 minutes per cycle and a total time of about 145minutes. The expected size of the amplification product was 404 bp.After reaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3% NuSieve 3:1 agarose. The results are shown in FIG.28A. FIG. 28A shows the results of electrophoresis for the detection ofenterohemorrhagic Escherichia coli O157 vero toxin II gene using theICAN or the PCR. Lane M1: molecular weight marker (50-2000 bp); lane M2:molecular weight marker (100 bp ladder); lane N: negative control; lane1: the template corresponding to 1 cell; lane 2: the templatecorresponding to 10 cells; lane 3: the template corresponding to 10²cells; lane 4: the template corresponding to 10³ cells; and lane 5: thetemplate corresponding to 10⁴ cells. Furthermore, results of comparisonbetween the amplification levels accomplished by the ICAN and the PCRfor 1 cell or 10 cells are shown in Table 10. TABLE 10 Number of E. coliO-157 cells 0 1 10 ICAN − + +++ PCR − + ++−: no amplification;+ to +++ indicate the degree of amplification in three grades.

As shown in FIG. 28A and Table 10, the amplification products ofinterest were obtained for the reaction systems using a hotwater-extract corresponding to 1 cell according to both the detectionmethod of the present invention and the PCR. Dot blot hybridization wasfurther carried out for the amplification product obtained according tothe ICAN using an oligonucleotide VT2 having a nucleotide sequencerepresented by SEQ ID NO:162 labeled with biotin at the 5′-terminus as aprobe. Hybridization was carried out under as follows. Briefly, 1 μl ofa reaction mixture was denatured at 98 C for 5 minutes, rapidly cooledon ice, and spotted onto Hybond-N membrane (Amersham Pharmacia Biotech).After exposure to UV, the membrane was placed in a hybridization bag. 10ml of a hybridization solution containing 0.5 M disodiumhydrogenphosphate (pH 7.2), 1 mM ethylenediaminetetraacetic acid and 7%sodium lauryl sulfate was added thereto. Pre-hybridization was thencarried out at 42 C for 30 minutes. 10 μl of the solution of the VT2probe at a concentration of 100 ng/μl was heat-denatured and added tothe pre-hybridization reaction system. After hybridization at 42 C for60 minutes, the membrane was washed twice in a solution containing 66.6mM sodium chloride, 66.6 mM sodium citrate and 0.1% sodium laurylsulfate at room temperature for 5 minutes, incubated in 6 ml of awashing buffer (0.3 M sodium chloride, 17.3 mM disodiumhydrogenphosphate dihydrate, 2.5 mM EDTA, 0.1% sodium lauryl sulfate) towhich 2 μl of 5 mg/ml horseradish peroxidase streptoavidin conjugate(PIERCE) was added at 42 C for 12 minutes, and then washed twice in thewashing buffer at room temperature. The membrane was then washed in 10ml of 0.1 M citrate buffer (pH 5.0) at room temperature and reacted in amixture of 5 ml of 0.1 M citrate buffer, 5 μl of 3% hydrogen peroxideand 250 μl of a solution of 2 mg/ml tetramethylbenzidine (TMB, NacalaiTesque) in ethanol in the dark for about 10 minutes. After colordevelopment, the reaction was terminated with deionized water. Theresults are shown in FIG. 28B. FIG. 28B shows the results of dot blothybridization for detecting a gene for vero toxin II fromenterohemorrhagic Escherichia coli O-157 by the ICAN. The results wereconsistent with those obtained for the above-mentioned electrophoresis.Thus, the detection sensitivity of the method of the present inventionwas equivalent to that of the PCR. The total time required for theamplification reaction using the ICAN of the present invention was ½ orshorter as compared with that required for the PCR. Thus, the ICAN ofthe present invention was confirmed to be effective as a method fordetecting a pathogen and the like.

Example 23

(1) Combination of a reverse transcription reaction and the method ofthe preset invention was examined using an RNA from cultured cells as atemplate. The reaction was carried out as follows. Briefly, RAW264.7cells (ATCC TIB 71) were suspended in Dulbecco's modified Eagle's medium(Bio Whittaker, 12-604F) containing 10% fetal calf serum (Gibco) at aconcentration of 1.5×10⁵ cells/ml. 5 ml of the suspension was added toeach well of a 6-well microtiter plate and the plate was incubated at 37C overnight in the presence of 5% CO₂. 50 μl of a 100 μg/ml solution oflipopolysaccharide (LPS, Sigma) in water and 50 μl of a 1000 U/μlsolution of interferon-γ (IFN-γ, Genzyme Techne) in water were added tothe well. The plate was incubated for additional 4 hours. An RNA wasthen prepared from the cells using RNeasy Mini Kit (Qiagen) according tothe instructions attached to the kit. As a negative control, a group towhich LPS or IFN-γ was not added was provided.

A cDNA was prepared by incubating 60 μl of a mixture containing 3 μg ofthe thus prepared RNA, 10 mM tris-hydrochloride buffer (pH 8.3), 50 mMKCl, 5 mM MgCl₂, 1 mM each of dNTPs, 150 pmol of random 6mers primer, 60U of ribonuclease inhibitor (Takara Shuzo) and 15 U of ReverseTranscriptase XL (AMV) (Takara Shuzo, 2620A) at 30 C for 10 minutes, 42C for 1 hour and then 99 C for 5 minutes for inactivating the enzymeusing a thermal cycler (GeneAmp PCR System 9600, Applied Biosystems).

Primers having nucleotide sequences represented by SEQ ID NOS:163 and164 were synthesized based on the nucleotide sequence of the mRNA formouse inducible NO synthase (iNOS) (GeneBank accession no. NM-010927).As a control, primers for PCR represented by SEQ ID NOS:165 and 166 werealso synthesized.

10 μl of a mixture containing 50 pmol each of the primers, 2 μl of anaqueous solution of propylenediamine at a concentration of 0.05%, 1 μlof the cDNA as a template (corresponding to 50 ng of the RNA) andsterile water was prepared. The mixture was heat-denatured at 98 C for 2minutes, cooled to 55 C and incubated at the temperature for 1 minute ina thermal cycler to anneal the primers to the template.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% bovine serum albumin (BSA), 1.25% dimethyl sulfoxide(DMSO), 15 U of E. coli RNase H and 11 U of BcaBEST DNA polymerase wasadded to the mixture to make the final volume to 50 μl with sterilewater. The reaction mixtures were incubated at 55 C for 1 hour in athermal cycler. The reacted samples were stored by freezing at −20 Cuntil being analyzed. The PCR as a control was carried out as follows.Briefly, 50 μl of a reaction mixture containing 50 pmol each of theprimers, 1 μl of the cDNA (corresponding to 50 ng of the RNA), 5 μl of10×Ex Taq buffer (Takara Shuzo), 1.25 U of TaKaRa Ex Taq DNA polymerase(Takara Shuzo) and 0.2 mM each of dNTPs was reacted in a thermal cycler.The program was as follows: 1 cycle of 94 C for 2 minutes; 30 cycles of94 C for 30 seconds, 55 C for 30 seconds and 72 C for 30 seconds; and 1cycle of 72 C for 5 minutes. The reacted samples were stored by freezingat −20 C until being analyzed. After reaction, 5 μl each of the reactionmixtures was subjected to electrophoresis on 3.0% agarose gel. Theresults are shown in FIG. 10.

FIG. 10 is a photograph of electrophoresis which shows the comparisonbetween the RT-ICAN and the RT-PCR. Lane 1: molecular weight marker (100bp ladder); lane 2: negative control group; and lane 3: a group treatedwith LPS and IFN-γ.

As shown in FIG. 10, the amplification products were observed for bothof the method of the present invention and the PCR only when the cDNAprepared from cells treated with LPS and IFN-γ was used as a template.Thus, it was confirmed that, since the method of the present inventionrequired a shorter period of time for reaction as compared with the PCR,the method of the present invention was more effective as a DNAamplification method after reverse transcription.

Example 24

E. coli RNase H of which the optimal temperature is 37 C may becomeinactivated during amplification reaction of the present invention.Then, the effect of addition of E. coli RNase H to the reaction mixtureduring the amplification reaction was examined. An amplified fragment(1071 bp) obtained by a PCR using primers GMO-PCR-F and GMO-PCR-Rrepresented by SEQ ID NOS:167 and 168 from a genomic DNA extracted fromrecombinant soybeans into which cauliflower mosaic virus ³⁵S promoterand EPSPS gene had been introduced was used as a template DNA. Inaddition, primers GMO-S1, S2, A1 and A2 having nucleotide sequencesrepresented by SEQ ID NOS:169 to 172 were used. The reaction was carriedout as follows. Briefly, 10 μl of a mixture containing 50 pmol each ofthe primers, propylenediamine at a final concentration of 0.01%, 1 pg to10 ng of the DNA as the template and sterile water was prepared. Themixture was heat-denatured at 98 C for 2 minutes and cooled to 55 C forannealing.

After annealing, at final concentrations, 500 μM each of dNTPs, 34 mMTricine-potassium hydroxide buffer (pH 8.7), 4.0 mM magnesium acetate,0.01% bovine serum albumin (BSA), 1% dimethyl sulfoxide (DMSO), 30 U ofE. coli RNase H and 5.5 U of BcaBEST DNA polymerase were added to themixture to make the final volume to 50 μl with sterile water. Thereaction mixtures were incubated at 55 C for 25 minutes in a thermalcycler. 30 U of E. coli RNase H was further added thereto 25 minutesafter the initiation of the reaction. The mixture was incubated at 55 Cfor 30 minutes. As a control, a reaction was carried out by incubatingthe mixture at 55 C for 55 minutes. After reaction, 3 μl each of thereaction mixtures was subjected to electrophoresis on 3% agarose. As aresult, it was confirmed that the amplification efficiency was improvedby the addition of E. coli RNase H during the reaction regardless of theconcentration of the template DNA for either of the combinations of theprimers, S1/A1, S1/A2, S2/A1 and S2/A2.

Example 25

The combination of a method for amplifying or duplicating a nucleic acidto be used as a template in the present invention and the method of thepresent invention was examined. The reaction was carried out as follows.Briefly, in vitro transcription was carried out using a plasmidcontaining the chrysanthemum viroid gene as prepared in Example 18(3)which was duplicated in Escherichia coli as a template and T7 RNApolymerase (Takara Shuzo) to obtain a fragment duplicated from an RNA. AcDNA was synthesized using primers having nucleotide sequencesrepresented by SEQ ID NOS:146 and 147 and cDNA synthesis kit (TakaraShuzo). An amplification reaction was carried out as described inExample 18(3) using the cDNA fragment or the duplicated plasmid as atemplate. As a result, it was confirmed that both a nucleic acidduplicated in a form of a plasmid and a nucleic acid in a form of a cDNAduplicated from an RNA using an RNA polymerase can be used as templatesfor the method of the present invention.

Example 26

(1) Synthesis of Primer

Oligonucleotide primers NS1 and NS2 represented by SEQ ID NOS:175 and176 were synthesized on the basis of the nucleotide sequence of the mRNAfor mouse inducible NO synthase (iNOS).

(2) Amplification of DNA Fragment According to ICAN Using PCR Product asTemplate

10 μl of a mixture containing 50 pmol each of the syntheticoligonucleotide primers, 2 μl of a 0.05% aqueous solution ofpropylenediamine and 10 fg to 10 pg of the template was heated at 98 Cfor 2 minutes and then 60 C for 2 minutes using a thermal cycler(GeneAmp PCR System 9600, Applied Biosystems) to anneal the primers tothe template. iNOS cDNA (741 bp) amplified using primers NS-PCR1 andNS-PCR2 represented by SEQ ID NOS:221 and 222, which was then purifiedusing Suprec02 (Takara Shuzo), was used as the DNA as the template. 40μl of a reaction mixture containing 0.625 mM each of dNTPs, 40 mMHEPES-potassium hydroxide buffer (pH 7.8), 125 mM potassium acetate, 5.0mM magnesium acetate, 0.0125% bovine serum albumin, 1.25% dimethylsulfoxide, 0.0156 pg of Pfu RNase H and 0.66 U of BcaBEST DNA polymerasewas added to the heated solution. The mixtures were at incubated at 60 Cfor 1 hour in a thermal cycler. 5 μl each of the reaction mixtures wasanalyzed by electrophoresis on 3.0% agarose gel. The results are shownin FIG. 11. FIG. 11 represents the results of the ICAN using Pfu RNaseH. Lane 1: molecular weight marker (100 bp); lane 2: 10 fg of thetemplate; lane 3: 100 fg of the template; lane 4: 1 pg of the template;and lane 5: 10 pg of the template.

As shown in FIG. 11, the amplification product of interest was observedusing 100 fg of the template.

Example 27

(1) Preparation of RNA

RAW264.7 cells (ATCC TIB 71) were suspended in Dulbecco's modifiedEagle's medium (Bio Whittaker) containing 10% fetal calf serum (Gibco)at a concentration of 1.5×10⁵ cells/ml. 5 ml of the suspension was addedto each well of a 6-well microtiter plate and the plate was incubated at37 C overnight in the presence of 5% CO₂. 50 μl of a 100 μg/ml solutionof lipopolysaccharide (LPS, Sigma) in water and 50 μl of a 1000 U/mlsolution of interferon-γ (IFN-γ, Genzyme Techne) in water were added tothe well. The plate was incubated for additional 4 hours. An RNA wasthen prepared from the cells using RNeasy Mini Kit (Qiagen, 74104)according to the instructions attached to the kit. As a negativecontrol, a group to which LPS or IFN-γ was not added was provided.

A cDNA was prepared by incubating 60 μl of a mixture containing 3 μg ofthe thus prepared RNA, 10 mM tris-hydrochloride buffer (pH 8.3), 50 mMKCl, 5 mM MgCl₂, 1 mM each of dNTPs, 150 pmol of random 6mers, 60 U ofRibonuclease Inhibitor (Takara Shuzo) and 15 U of Reverse TranscriptaseXL (AMV) (Takara Shuzo) at 30 C for 10 minutes, 42 C for 1 hour and then99 C for 5 minutes for inactivating the enzyme using a thermal cycler(GeneAmp PCR System 9600, Applied Biosystems).

Primers NS5 and NS6 having nucleotide sequences represented by SEQ IDNOS:181 and 182 were synthesized based on the nucleotide sequence of themRNA for mouse inducible NO synthase (iNOS). Furthermore, primers NS3and NS4 for PCR represented by SEQ ID NOS:177 and 178 were alsosynthesized.

50 μl of a mixture containing 50 pmol each of the primers NS5 and NS6, 1μl of the cDNA solution synthesized as described above (corresponding to50 ng of the RNA) or a 10-, 100-, 1000- or 10000-fold dilution withwater thereof as a template, 0.5 mM each of dNTPs, 32 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 4.0 mM magnesiumacetate, 0.01% bovine serum albumin, 1% dimethyl sulfoxide, 0.0156 pg ofPfu RNase H and 0.66 U of BcaBEST DNA polymerase was incubated at 60 Cfor 1 hour in a thermal cycler. The reacted samples were stored byfreezing at −20 C until being analyzed.

On the other hand, a PCR was carried out as a control. 50 μl of areaction system containing 50 pmol each of the primers NS3 and NS4, 1 μlof the cDNA solution (corresponding to 50 ng of the RNA) or a 10-, 100-,1000- or 10000-fold dilution with water thereof, 5 μl of 10×Ex Taqbuffer (Takara Shuzo), 1.25 U of TaKaRa Ex Taq polymerase (Takara Shuzo)and 0.2 mM each of dNTPs was reacted using a thermal cycler. The programwas as follows: 1 cycle of 94 C for 2 minutes; 35 cycles of 94 C for 30seconds, 55 C for 30 seconds and 72 C for 30 seconds; and 1 cycle of 72C for 5 minutes. The reacted samples were stored by freezing at −20 Cuntil being analyzed.

5 μl each of the reaction mixtures (ICAN or PCR) was analyzed byelectrophoresis on 3.0% agarose gel. The results are shown in FIG. 12.FIG. 12 shows the results of the detection of iNOS gene according to theICAN using Pfu RNase H or the PCR. Lane 1: a lane for 100 bp DNA laddermarker; lane 2: the 10000-fold dilution of the negative control cDNA;lane 3: the 1000-fold dilution of the negative control cDNA; lane 4: the100-fold dilution of the negative control cDNA; lane 5: the 10-folddilution of the negative control cDNA; lane 6: the original solution ofthe negative control cDNA; lane 7: the 10000-fold dilution of the cDNAfrom the group with the addition of LPS and IFN-γ; lane 8: the 1000-folddilution of the cDNA from the group with the addition of LPS and IFN-γ;lane 9: the 100-fold dilution of the cDNA from the group with theaddition of LPS and IFN-γ; lane 10: the 10-fold dilution of the cDNAfrom the group with the addition of LPS and IFN-γ; and lane 11: theoriginal solution of the cDNA from the group with the addition of LPSand IFN-γ.

As shown in FIG. 12, the amplification products were observed for bothof the ICAN and the PCR only when the cDNA prepared from cells treatedwith LPS and IFN-γ was used as a template. For the ICAN, the increase inamplification product was observed using the 1000-fold dilution of thecDNA. For the PCR, the increase in amplification product was observedusing the 100-fold dilution of the cDNA.

Example 28

(1) Oligonucleotide primers 4 and 5 represented by SEQ ID NOS:179 and180 were synthesized based on the nucleotide sequence of λ DNA. Theoligonucleotide primer 4 is a sense primer having 75% of a GC content.The oligonucleotide primer 5 is an antisense primer having 80% of a GCcontent.

10 μl of a reaction system containing 120 pmol each of the primers 4 and5, 2 μl of a 0.05% propylenediamine solution and 10 ng of a template washeat-denatured at 98 C for 2 minutes, and then rapidly cooled on ice toanneal the primers to the template. The PCR product (1005 bp) purifiedusing Suprec02 as described in Example 15 was used as the template.

(2) After annealing, 40 μl of a mixture containing 0.625 mM each ofdNTPs, 42.5 mM Bicine-potassium hydroxide buffer (pH 8.3), 5.0 mMmagnesium acetate, 0.0125% bovine serum albumin, 1.25% dimethylsulfoxide, 0.5 μl of Thermotoga maritima RNase HII (0.58 μg/ml) and 2.2U of BcaBEST DNA polymerase was added to the mixture to carry out theICAN at 60, 65 or 70 C for 1 hour. 3 μl each of the reaction mixturesafter the ICAN was confirmed by electrophoresis on 3.0% agarose gel. Theresults are shown in FIG. 13. FIG. 13 shows the results of the ICANusing Thermotoga maritima RNase HII. Lane 1: molecular weight marker(100 bp); lane 2: reaction temperature of 60 C; lane 3: reactiontemperature of 65 C; and lane 4: reaction temperature of 70 C.

As shown in FIG. 13, the amplification products of interest wereobserved for the respective reaction temperatures.

Example 29

(1) Amplification of a DNA fragment according to the ICAN using a PCRproduct as a template (alkali-denatured) was examined. 1 μl of asolution containing 10 fg to 10 pg of a template and 1 μl of 0.4 N NaOHwere mixed together. The mixture was incubated at 37 C for 5 minutes todenature the template. The PCR-amplified iNOS cDNA (741 bp) purifiedusing Suprec02 (Takara Shuzo) as described in Example 26 was used as atemplate. Each of the denatured templates was neutralized using 1 μl of0.4 N HCl. 47 μl of a reaction mixture containing 50 pmol each of theprimers NS1 and NS2, 0.5 mM each of dNTPs, 32 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 4.0 mM magnesiumacetate, 0.01% bovine serum albumin, 1.0% dimethyl sulfoxide, 0.0156 pgof Pfu RNase H and 0.66 U of BcaBEST DNA polymerase was then addedthereto. The mixture was incubated at 60 C for 1 hour in a thermalcycler. 5 μl each of the reaction mixtures was analyzed byelectrophoresis on 3.0% agarose gel. The results are shown in FIG. 14.FIG. 14 shows the results of the ICAN using an alkali-denaturedtemplate. Lane 1: molecular weight marker (100 bp); lane 2: 10 fg of thetemplate; lane 3: 100 fg of the template; lane 4: 1 pg of the template;and lane 5: 10 pg of the template.

As shown in FIG. 14, the amplification product was clearly increasedusing 1 pg of the template.

Example 30

(1) Amplification of a DNA fragment according to the ICAN withoutdenaturing a template was examined. Primers NS5 and NS6 represented bySEQ ID NOS:181 and 182 were used as primers. The template as prepared inExample 26 was used as a template DNA.

50 μl of a reaction mixture containing 10 fg to 100 pg of the templateor water for a negative control, 50 pmol each of the primers NS5 andNS6, 0.5 mM each of dNTPs, 32 mM HEPES-potassium hydroxide buffer (pH7.8), 100 mM potassium acetate, 4.0 mM magnesium acetate, 0.01% bovineserum albumin, 1.0% dimethyl sulfoxide, 0.0156 pg of Pfu RNase H and 1 Uof BcaBEST DNA polymerase (Takara Shuzo) was incubated at 60 C for 1hour in a thermal cycler. After reaction, 5 μl each of the reactionmixture was analyzed by electrophoresis on 3.0% agarose gel. Aphotograph of the electrophoresis is shown in FIG. 15. FIG. 15 is aphotograph of electrophoresis for the amplification method of thepresent invention without denaturing a template DNA. Lane 1: 100 bp DNAladder marker; lane 2: negative control (water); lane 3: 10 fg of thetemplate; lane 4: 100 fg of the template; lane 5: 1 pg of the template;lane 6: 10 pg of the template; and lane 7: 100 pg of the template.

As shown in FIG. 15, the amplification product of interest was observedusing 1 pg of the template.

Example 31

(1) Primers pDON-AI-1 and pDON-AI-2 represented by SEQ ID NOS:183 and184 were synthesized based the nucleotide sequence of the packagingregion in a vector plasmid pDON-AI DNA (Takara Shuzo).

(2) Amplification of DNA Fragment According to ICAN Without DenaturingTemplate

50 μl of a reaction mixture containing 1 μl of a solution containing 10fg to 1 ng of pDON-AI DNA or water for a negative control, 50 pmol eachof the primers, 0.5 mM each of dNTPs, 32 mM HEPES-potassium hydroxidebuffer (pH 7.8), 100 mM potassium acetate, 4.0 mM magnesium acetate,0.01% bovine serum albumin, 1.0% dimethyl sulfoxide, 0.0156 pg of PfuRNase H as prepared in Referential Example 4 and 1 U of BcaBEST DNApolymerase was incubated at 60 C for 1 hour in a thermal cycler. 5 μleach of the reaction mixtures was analyzed by electrophoresis on 3.0%agarose gel. The results are shown in FIG. 16. FIG. 16 is a photographof electrophoresis for the method of the present invention using acircular double-stranded DNA as a template without denaturation. Lane 1:100 bp DNA ladder marker; lane 2: negative control (water); lane 3: 10fg of the template; lane 4: 100 fg of the template; lane 5: 1 pg of thetemplate; lane 6: 10 pg of the template; lane 7: 100 pg of the template;and lane 8: 1 ng of the template.

As shown in FIG. 16, it was confirmed that the amplification product ofinterest was obtained using 10 fg of the template.

Example 32

Detection of human papilloma virus 16 gene utilizing the method of thepresent invention was examined. A DNA from cells infected with humanpapilloma virus 16, CaSki cells (Dainippon Pharmaceutical; containing500 copies of human papilloma virus 16 in a cell), was used as atemplate. Primers HPV16 S3 and HPV16 A2 having nucleotide sequencesrepresented by SEQ ID NOS:185 and 186 were used as primers for detectingHPV16. The expected size of the amplification product obtained using theprimer pair was about 120 bp. The reaction was carried out as follows.

10 μl of a mixture containing 1 pg, 3 pg, 30 pg, 100 pg, 300 pg, 1 ng, 3ng or 10 ng of the template DNA, 50 pmol each of the primers HPV16 S3and HPV16 A2 and propylenediamine at a final concentration of 0.01% wasprepared. The mixtures were incubated at 98 C for 2 minutes and at 55 Cfor 1 minute in Thermal Cycler Personal, and then placed on ice. Atfinal concentrations, 20 mM HEPES-potassium hydroxide buffer (pH 7.8),100 mM potassium acetate, 1% dimethyl sulfoxide, 0.01% bovine serumalbumin, 4 mM magnesium acetate, 500 μM each of dNTPs, 30 U of E. coliRNase H and 5.5 U of BcaBEST DNA polymerase were added to the mixture tomake the final volume to 50 μl. The reaction mixtures were placed in athermal cycler which had been set at 55 C and reacted for 60 minutes. Asa control, a PCR was carried out using Human Papillomavirus PrimersHPVp16 (forward, reverse) (Takara Shuzo) according to the manual inThermal Cycler Personal. The expected size of the amplification productwas 140 bp.

After reaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 4% NuSieve 3:1 agarose. The results are shown in FIG.17A. FIG. 17A shows the results of the detection of HPV16 gene utilizingthe ICAN and the PCR. Lane M1: molecular weight marker (100 bp ladder);lane M2: molecular weight marker (50-2000 bp); lane 1: no template; lane2: 1 pg of the template; lane 3: 3 pg of the template; lane 4: 30 pg ofthe template; lane 5: 100 pg of the template; lane 6: 300 pg of thetemplate; lane 7: 1 ng of the template; lane 8: 3 ng of the template;and lane 9: 10 ng of the template.

As shown in FIG. 17A, it was confirmed that the amplification productsof interest were obtained using 3 pg of the template DNA for the ICANand 1 pg of the template DNA for the PCR, respectively.

Furthermore, dot blot hybridization for the reaction products wascarried out using an oligonucleotide HPV16 probe having a nucleotidesequence represented by SEQ ID NO:187. Hybridization was carried out asdescribed in Example 22. The results are shown in FIG. 17B. FIG. 17Bshows the results of dot blot hybridization detection of HPV16 geneaccording to the PCR and the ICAN. Lane 1: no template; lane 2: 1 pg ofthe template; lane 3: 3 pg of the template; lane 4: 30 pg of thetemplate; lane 5: 100 pg of the template; lane 6: 300 pg of thetemplate; lane 7: 1 ng of the template; lane 8: 3 ng of the template;and lane 9: 10 ng of the template.

As shown in FIG. 17B, the detection sensitivities of the ICAN and thePCR were almost equivalent. Thus, it was confirmed that these methodswere effective for detecting a virus or the like.

Example 33

Detection of human papilloma virus 16 gene from a clinical specimen DNAsample was examined. DNAs prepared according to a conventional methodfrom 6 clinical specimens obtained with informed consent were used astemplates. The types of the infecting HPVs in the samples prepared fromthese clinical specimens had been proved by the PCR. The primers HPV16S3 and HPV16 A2 as described in Example 32 were used as primers fordetection. The concentrations of the DNA samples from the clinicalspecimens to be used as templates were adjusted to 100 ng/μl with TEbuffer. The composition of the reaction mixture and the reactionconditions as described in Example 32 were used except for the amount ofthe template. In addition, similar reactions were carried out using areaction mixture without the addition of the template DNA as a negativecontrol and a reaction mixture containing 500 pg of the DNA from CaSkicells infected with HPV 16 as a positive control. After reaction, 3 μleach of the reaction mixtures was subjected to electrophoresis on 4%NuSieve 3:1 agarose. The results are shown in FIG. 18A. FIG. 18A showsthe results of the detection of HPV16 gene from clinical specimens. LaneM: molecular weight marker; lanes 1 to 6: clinical specimens; lane 7:negative control; and lane 8: positive control.

As shown in FIG. 18A, the amplification products of about 120 bp wereobserved according to the ICAN for the samples which had been proved tobe infected with HPV16 by the conventional PCR. No amplification wasobserved for samples infected with other types of HPVs or non-infectedsamples.

Furthermore, dot blot hybridization for the amplification products wascarried out as described in Example 22. The results are shown in FIG.18B and Table 11. FIG. 18B shows the results of dot blot hybridizationdetection of HPV16 gene from the clinical specimens. Lanes 1 to 6:clinical specimens; lane 7: negative control; and lane 8: positivecontrol.

As shown in FIG. 18B, results consistent with those obtained by theelectrophoresis were obtained, confirming that similar results withthose of the PCR could be obtained by using electrophoresis as well asdot blot hybridization. Thus, it was confirmed that HPV16 could bedetected from practical clinical specimens according to the method ofthe present invention and that the method was effective for detecting avirus or the like. TABLE 11 No Positive Sample No. 3 No. 4 No. 6 No. 7No. 8 No. 9 template control Typing by non- non- Type Type Type Type * *PCR infected infected 18 16 67 16 ICAN − − − + − + − + amplificationusing HPV16 detection primer−: no amplification;+: amplification observed.

Example 34

Detection of HCV from clinical specimens was examined. Specimen sampleswere prepared from 300 μl each of 5 serum specimens from patients withHCV obtained with informed consent using TRIzol reagent (LifeTechnologies) according to the instructions attached to the reagent andfinally dissolved in 6 μl of injectable water (Otsuka Pharmaceutical) toobtain RNA samples. An RNA similarly extracted from 300 μl of a serumfrom a healthy individual was used as a negative control. First, 4 μl ofa reaction mixture for reverse transcription containing 1×RNA PCRBuffer, 5 mM MgCl₂, 1 mM dNTPs, 1 U of AMV Reverse Transcriptase XL, 10pmol each of primers HCV-F and HCV-R represented by SEQ ID NOS:188 and189, and 2 μl of one the RNA samples was prepared using RNA PCR kit(AMW) ver 2.1 (Takara Shuzo). The mixtures were warmed at 30 C for 10minutes and then reacted at 50 C for 30 minutes. After reversetranscription, the ICAN was carried out. Primers HCV-F2 and HCV-R1having nucleotide sequences represented by SEQ ID NOS:190 and 191 wereused for the ICAN. The reaction was carried as follows.

10 μl of a mixture containing 50 pmol each of the primers, 3 μl one ofthe reverse transcription reaction mixtures and propylenediamine at afinal concentration of 0.01% was prepared. 3 μl of sterile water wasused for a blank. The mixtures were heated at 98 C for 2 minutes,rapidly cooled to 60 C and incubated at the temperature for 1 minute inThermal Cycler Personal, and stored on ice.

After annealing, at final concentrations, 20 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 1% dimethylsulfoxide, 0.01% bovine serum albumin, 4 mM magnesium acetate, 500 μMeach of dNTPs, 30 U of E. coli RNase H and 5.5 U of BcaBEST DNApolymerase were added to the mixture to make the final volume to 50 μlwith sterile water. The reaction mixtures were placed in Thermal CyclerMP which had been set at 60 C and reacted for 60 minutes. Afterreaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3% NuSieve 3:1 agarose. The results are shown in FIG.19A. FIG. 19A shows the results of HCV detection from clinicalspecimens. Lane B: sterile water as a template; lane 1: a sample from ahealthy individual; lanes 2 to 6: samples from patients with HCV; andlane M: molecular weight marker (50-2000 bp).

As shown in FIG. 19A, the amplification products of about 107 bpexpected from the nucleotide sequence of HCV genome were observed onlyfor the RNA samples from patients with HCV, whereas such anamplification product was not observed for the serum from the healthyindividual and the blank. Furthermore, dot blot hybridization for theICAN-amplified products was carried out as described in Example 22 usinga probe for HCV represented by SEQ ID NO:192 biotinated at 5′-terminus.The results are shown in FIG. 19B. The lanes for the samples in FIG. 19Bare as those indicated for the photograph of electrophoresis.

As shown in FIG. 19, it was confirmed that the results from theelectrophoresis were consistent with those from the dot blothybridization. These results confirms that the method of the presentinvention can be used to detect HCV from practical clinical specimensand is effective for detecting a virus or the like.

Example 35

A method for detecting adenovirus was examined.

Primers for amplifying E1A (tumor gene), E1A-1 (sense), E1A-2(antisense) and E1A-3 (antisense), represented by SEQ ID NOS:193 to 195were constructed based on the nucleotide sequence of adenovirus (GenBankaccession no. J01917). Adenovirus (ATCC accession no. VR-5) was used. Atemplate was prepared as follows. 100 μl of a solution containingadenovirus at a concentration of 8.73×10¹⁰ PFU/ml was incubated in thepresence of SDS at a final concentration of 0.1% and proteinase K at afinal concentration of 0.2 mg/ml at 37 C for 1 hour. The DNA waspurified by affinity to silica gel. Adenoviral DNAs corresponding to10³, 10⁴, 105 or 10⁶ PFU prepared by diluting the purified DNA withsterile water were used. The reaction was carried out as follows.Briefly, 10 μl of a reaction system containing 60 pmol each of theprimers E1A-1 and E1A-2 (chain length to be amplified: 112 bp) or E1A-1and E1A-3 (chain length to be amplified: 91 bp), 2 μl of 0.05%propylenediamine and the template was heat-denature at 98 C for 2minutes and then rapidly cooled on ice to anneal the primers to thetemplate.

After annealing, 40 μl of a mixture containing 0.625 mM each of dNTPs,42.5 mM Tricine-potassium hydroxide buffer (pH 8.5), 5.0 mM magnesiumacetate, 0.0125% bovine serum albumin, 1.25% dimethyl sulfoxide, 30 U ofE. coli RNase H and 5.5 U of BcaBEST DNA polymerase was added to themixture to make the final volume to 50 μl. The reaction mixtures wereincubated at 60 C for 1 hour. As a control, a detection by a PCR wascarried out using the same template as the above and primers constructedfor PCR-amplifying E1A (tumor gene), E1A-1P (sense), E1A-2P (antisense)and E1A-3P (antisense) having nucleotide sequences represented by SEQ IDNOS:196, 197 and 231. The PCR was carried out as follows. Brief ly, 50μl of a PCR solution containing 60 pmol each of the primers E1A-1P andE1A-2P (chain length to be amplified: 112 bp) or E1A-1P and E1A-3P(chain length to be amplified: 91 bp), 5 μl of 10×Ex Taq buffer (TakaraShuzo), 1.25 U of TaKaRa Ex Taq DNA polymerase (Takara Shuzo) and 0.2 mMeach of dNTPs was prepared. The conditions for the PCR were as follows:30 cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 C for 30seconds.

After reaction, 3 μl each of the reaction mixtures of the ICAN and thePCR was subjected to electrophoresis on 3.0% agarose gel. The resultsare shown in FIG. 20 and Table 12. FIG. 20 shows the results of thedetection of viral E1A gene from adenoviral particles. Lanes 1 to 10show the results obtained using the combination of the primers E1A-1 andE1A-2. Lanes 11 to 20 show the results obtained using the combination ofthe primers E1A-1 and E1A-3. Lane 1: molecular weight marker (100 bpladder); lane 2: ICAN using DNA corresponding to 10⁶ PFU; lane 3: ICANusing DNA corresponding to 10⁵ PFU; lane 4: ICAN using DNA correspondingto 104 PFU; lane 5: ICAN using DNA corresponding to 10³ PFU; lane 6:molecular weight marker (100 bp ladder); lane 7: PCR using DNAcorresponding to 10⁶ PFU; lane 8: PCR using DNA corresponding to 10⁵PFU; lane 9: PCR using DNA corresponding to 10⁴ PFU; and lane 10: PCRusing DNA corresponding to 10³ PFU. In addition, lane 11: molecularweight marker (100 bp ladder); lane 12: ICAN using DNA corresponding to10⁶ PFU; lane 13: ICAN using DNA corresponding to 10⁵ PFU; lane 14: ICANusing DNA corresponding to 10⁴ PFU; lane 15: ICAN using DNAcorresponding to 10³ PFU; lane 16: molecular weight marker (100 bpladder); lane 17: PCR using DNA corresponding to 10⁶ PFU; lane 18: PCRusing DNA corresponding to 10⁵ PFU; lane 19: PCR using DNA correspondingto 104 PFU; and lane 20: PCR using DNA corresponding to 10³ PFU. TABLE12 Amplification size Detection limit (bp) ICAN PCR 112 10⁴ 10⁴ 91 10⁴10⁴

As shown in FIG. 20 and Table 12, it was confirmed that the detectionsensitivity for the detection of adenovirus E1A gene by the ICAN wasequivalent to that by the PCR.

Example 36

Detection of an integrated viral gene from cells infected with aretrovirus vector was examined. Cells infected with a retrovirus and agenomic DNA were prepared as follows. Briefly, a vector plasmid pDON-AI(Takara Shuzo) was introduced into packaging cells GPE+86 according tothe calcium phosphate method. An ecotropic vector was prepared from theculture supernatant of the introduced cells. Cells infected with a virusvector were prepared by infecting NIH/3T3 cells with the ecotropicvector and culturing the infected cells for 14 days in a mediumcontaining G418. 27 μg of a genomic DNA was obtained according to aconventional method from 4×10⁴ of the prepared cells infected with theretrovirus. The primers pDON-AI-1 and pDON-AI-2 as described in Example31(1) were used as primers. The reaction was carried out as follows.Briefly, 10 μl of a reaction system containing 60 pmol each of theprimers, 2 μl of a 0.25% aqueous solution of propylenediamine and 0.1 ngto 1000 ng of the genomic DNA as a template was heated at 98 C for 2minutes and then at 60 C in a thermal cycler (Takara Shuzo) to annealthe primers to the template.

After annealing, 40 μl of a reaction mixture containing 0.625 mM each ofdNTPs, 40 mM HEPES-potassium hydroxide buffer (pH 7.8), 125 mM potassiumacetate, 5 mM magnesium acetate, 0.0125% bovine serum albumin, 1.25%dimethyl sulfoxide, 30 U of E. coli RNase H and 5.5 U of BcaBEST DNApolymerase was added to the mixture to make the final volume to 50 μl.The reaction mixtures were incubated at 60 C for 1 hour in a thermalcycler. After reaction, 5 μl each of the reaction mixtures was subjectedto electrophoresis on 3.0% agarose gel. In addition, a PCR was carriedout using primers pDON-AI-3 and pDON-AI-4 represented by SEQ ID NOS:200and 201 in order to compare the sensitivities of detecting a DNA by theICAN and the PCR. The PCR was carried out as follows. 50 μl of areaction mixture containing 0.1 ng to 100 ng of the template, 60 pmoleach of the primers, 5 μl of 10×Ex Taq buffer, 1.25 U of TaKaRa Ex Taqpolymerase and 0.2 mM each of dNTPs was prepared. The mixtures weresubjected to reactions using Thermal Cycler Personal as follows: 35cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 C for 30seconds. After reaction, 5 μl each of the reaction mixtures wassubjected to electrophoresis on 3.0% agarose gel. The results are shownin FIG. 21. FIG. 21 shows the results of the detection of an integratedviral gene from cells infected with a retrovirus vector according to theICAN and the PCR. Lane 1: molecular weight marker (100 bp ladder); lane2: 1000 ng of the template; lane 3: 100 ng of the template; lane 4: 10ng of the template; lane 5: 1 ng of the template; and lane 6: 0.1 ng ofthe template.

As shown in FIG. 21, the amplification products of interest wereobserved for the ICAN using 1 ng of the template DNA and for the PCR of35 cycles using 1 ng of the template.

Example 37

A method for detecting a target nucleic acid in which the amplificationmethod of the present invention and a hybridization method were combinedwas examined for the detection of Escherichia coli O-157 vero toxin Igene. Vero toxin I gene from enterohemorrhagic Escherichia coli O-157was selected as a target. The template DNA was prepared as described inExample 21(1). A region of about 80 bp having a GC content of about 40%was selected as a region to be amplified. Primers VT1-IF4 and VT1-IR1having nucleotide sequences represented by SEQ ID NOS:202 and 203 wereused as primers. The reaction was carried out as follows. Briefly, 5 μlof a mixture containing 60 pmol each of the primers VT1-IF4 and VT1-IR1,propylenediamine at a final concentration of 0.01%, one of hotwater-extracts corresponding to 0 to 10⁵ cells and sterile water wasprepared. The mixtures were heat-denatured at 98 C for 2 minutes,rapidly cooled to 55 C and incubated at the temperature for 1 minute inThermal Cycler Personal, and then placed on ice for annealing.

After annealing, at final concentrations, 20 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 1% dimethylsulfoxide, 0.01% bovine serum albumin, 4 mM magnesium acetate, 500 μMeach of dNTPs, 15 U of E. coli RNase H and 2.75 U of BcaBEST DNApolymerase were added to the mixture to make the final volume to 25 μlwith sterile water. The reaction mixtures were placed in Thermal CyclerPersonal which had been set at 55 C and incubated at the temperature for60 minutes. As a control, a PCR for the hot water-extract was carriedout using O-157 Typing Set (Takara Shuzo) according to the manual inThermal Cycler Personal. The PCR was carried out as follows: 35 cyclesof 94 C for 1 minute, 55 C for 1 minute and 72 C for 1 minute. The totaltime required for the reaction was about 145 minutes. The expected sizeof the amplification product was 349 bp. After reaction, 3 μl each ofthe reaction mixtures was subjected to electrophoresis on 3% NuSieve 3:1agarose. The results for the ICAN are shown in FIG. 22. FIG. 22 showsthe results of the detection of O-157 vero toxin I gene. Lane M:molecular weight marker (50-2000 bp); lane N: sterile water as template;lane 1: the template corresponding to 1 cell; lane 2: the templatecorresponding to 10 cells; lane 3: the template corresponding to 10²cells; and lane 4: the template corresponding to 10³ cells. Furthermore,the results of the detection by the ICAN and the PCR are shown in Table13. TABLE 13 Number of Escherichia coli O-157 cells 0 1 10 ICAN − + +++PCR − + ++−: no amplification;+ to +++ indicate the degree of amplification in three grades.

As shown in Table 13, the amplification products of interest wereobtained for the reaction systems in which the hot water-extractcorresponding to 1 cell was used for both of the ICAN and the PCR.Furthermore, dot blot hybridization for the amplification products wascarried out using an oligonucleotide probe VT1 having a nucleotidesequence represented by SEQ ID NO:204 labeled with biotin at the5′-terminus. The hybridization was carried out as described in Example32. The results were consistent with those obtained by theelectrophoresis. Thus, it was confirmed that the detection sensitivitiesof the ICAN and the PCR were equivalent. In addition, the total timerequired for the amplification reaction using the ICAN of the presentinvention was ½ or shorter as compared with that required for the PCR.Thus, the ICAN of the present invention was confirmed to be effective asa method for detecting a pathogenic bacterium and the like.

Example 38

A method for detecting a gene for botulinum toxin type A was examined. ADNA prepared from Clostridium botulinum, a strain from a food poisoningcase, type A-190 was used as a template. This strain is preserved inDepartment of Hygiene, Kagawa Nutrition University. Primers BotA S2 andBotA A2 having nucleotide sequences represented by SEQ IOD NOS:205 and206 were synthesized as primers for detection. The expected size of theamplification product obtained using the primer pair was about 150 bp.Solutions containing 100 fg, 1 pg, 10 pg or 100 pg of the DNA from typeA toxin-producing Clostridium botulinum in 1 μl of sterile water to beused as templates were prepared. The reaction was carried out asfollows.

10 μl of a mixture containing 50 pmol each of the primers,propylenediamine at a final concentration of 0.01% and 1 μl of one ofthe DNA solutions as a template was prepared. The mixtures weresubjected to the ICAN using the composition of the reaction mixture andthe reaction conditions as described in Example 32. As a control, a PCRwas carried out in using a primer set for detecting botulinum toxin typeA gene, BAS-1 and BAS-2 (Takara Shuzo), according to the manual inThermal Cycler Personal. The expected size of the amplification productwas 284 bp.

After reaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 4% NuSieve 3:1 agarose. The results are shown in FIG.23A. FIG. 23A shows the results of the detection of the gene forbotulinum toxin type A according to the ICAN and the PCR. Lane M1:molecular weight marker (100 bp ladder); lane M2: molecular weightmarker (50-2000 bp marker); lane 1: no template; lane 2: 100 fg of thetemplate; lane 3: 10 pg of the template; and lane 4: 100 pg of thetemplate.

As shown in FIG. 23A, the amplification product of interest was observedfor the reaction in which 100 fg of the template DNA was used accordingto the ICAN. On the other hand, the amplification product of interestwas not observed for the reaction in which 100 fg of the template DNAwas used according to the PCR. Furthermore, dot blot hybridization forthe reaction products was carried out using BotA probe having anucleotide sequence represented by SEQ ID NO:207. Dot blot hybridizationwas carried out as described in Example 22. The results are shown inFIG. 23B. As shown in FIG. 23B, the signals were observed using 100 fgof the template for the ICAN and 10 pg of the template for the PCR,respectively. These results were consistent with those of theelectrophoresis.

Example 39

Detection of chrysanthemum viroid was examined. 10-fold serial dilutionsof low molecular weight RNA obtained according to the method forextracting low molecular weight RNAs from chrysanthemum infected withchrysanthemum stunt viroid (CSVd) as described in Example 1 of JP-A9-140383 were prepared. A reverse transcription reaction was carried outusing RNA PCR kit (AMW) ver 2.1 (Takara Shuzo). Specifically, 20 μl of areaction mixture for reverse transcription containing 1×RNA PCR Buffer,5 mM MgCl₂, 1 mM each of dNTPs, 20 U of RNase Inhibitor, 5 U of AMVReverse Transcriptase XL, 50 pmol of Random 9mers, 1 μl of one of theserial dilutions of the RNA was prepared. The mixtures were warmed at 30C for 10 minutes, reacted at 55 C for 30 minutes and then heated at 99 Cfor 5 minutes for inactivating the reverse transcriptase. After cooling,the ICAN was carried out. 1 μl of the reverse transcription reactionmixture was used as a template for 50 μl of a reaction mixture for theICAN. Primers CSVD-F4 and CSVD-R3 having nucleotide sequencesrepresented by SEQ ID NOS:208 and 209 were used as primers in thisExample. The reaction was carried out as described in Example 32 exceptthat reaction temperature was 60 C and that Thermal Cycler MP was used.After reaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3% NuSieve 3:1 agarose.

On the other hand, PCR amplification was carried out in a reactionsystem of 50 μl using 1 μl of the same reverse transcription reactionmixture as a template. Primers F94 and R264 represented by SEQ IDNOS:198 and 199 were used as primers. The reaction was carried out asfollows. Briefly, a reaction mixture was prepared using TaKaRa PCRAmplification kit according to the protocol. 10 pmol each of the primersand 1 μl of one of the reverse transcription reaction mixture were addedthereto to make the total volume to 50 μl. An amplification reaction wascarried out using Thermal Cycler MP. The reaction was carried out asfollows: 30 cycles of 94 C for 30 seconds, 55 C for 30 seconds and 72 Cfor 30 seconds. After reaction, 5 μl each of the reaction mixtures wassubjected to electrophoresis on 3% NuSieve 3:1 agarose. The results areshown in Table 14. TABLE 14 Dilution rate of RNA as template 10² 10³RT-ICAN ++ + RT-PCR + −−: not amplified;+: amplified;++: amplified well.

As shown in Table 14, the amplification product was observed for thereaction in which the 10³-fold-diluted RNA sample was used as a templateaccording to the ICAN. On the other hand, the amplification product wasobserved for the reaction in which the 10²-fold-diluted RNA sample wasused as a template according to the PCR.

Furthermore, the ICAN-amplified products and the PCR-amplified productswere confirmed to be the products of interest by dot blot hybridization.Dot blot hybridization was carried out using CSVD probe having anucleotide sequence represented by SEQ ID NO:210 labeled with biotin atthe 5′-terminus. Hybridization was carried out as described in Example22. The results were consistent with those of the electrophoresis. Thesignals were observed using the 10³-fold-diluted RNA sample for the ICANand the 10²-fold-diluted RNA sample for the PCR, respectively,demonstrating that the ICAN was more sensitive than the PCR.

Example 40

Detection of a viroid gene from chrysanthemum infected withchrysanthemum dwarfing viroid (CSVd) using Pfu RNase H was examined. 60μl of a mixture containing 3 μl of one of the 10-fold serial dilutionsof the RNA as prepared in Example 39, 10 mM tris-hydrochloride buffer(pH 8.3), 50 mM potassium chloride, 5 mM magnesium chloride, 1 mM eachof dNTPs, 150 pmol of Random 6mers, 60 U of Ribonuclease Inhibitor(Takara Shuzo) and 15 U of Reverse Transcriptase XL (AMV) (Takara Shuzo)was incubated at 30 C for 10 minutes and at 42 C for 1 hour and thenheated at 99 C for 5 minutes for inactivating the enzyme using a thermalcycler (GeneAmp PCR System 9600, Applied Biosystems) to prepare a cDNA.Primers Vd1, Vd2, Vd3 and Vd4 represented by SEQ ID NOS:211 to 214 weresynthesized based on the nucleotide sequence of the mRNA for the viroid.

50 μl of a mixture containing 50 pmol each of the primers Vd1 and Vd2, 1μl of the cDNA solution synthesized as described above, a 10-, 100-,1000- or 10000-fold dilution thereof with water or water for a negativecontrol as a template, 0.5 mM each of dNTPs, 32 mM HEPES-potassiumhydroxide buffer (pH 7.8), 100 mM potassium acetate, 4.0 mM magnesiumacetate, 0.01% bovine serum albumin, 1% dimethyl sulfoxide, 0.0156 μg ofPfu RNase H and 1 U of BcaBEST DNA polymerase was incubated at 57 C for1 hour in a thermal cycler. The reacted samples were stored by freezingat −20 C until being analyzed.

A PCR was carried out as a control. Briefly, 50 μl of a reaction systemcontaining 50 pmol each of the primers Vd3 and Vd4, 1 μl of the cDNAsolution, a 10-, 100-, 1000- or 10000-fold dilution thereof with wateror water for a negative control, 5 μl of 10×Ex Taq buffer, 1.25 U ofTaKaRa Ex Taq polymerase and 0.2 mM each of dNTPs was subjected to areaction in a thermal cycler. The program was as follows: 1 cycle of 94C for 2 minute; 35 cycles of 94 C for 30 seconds, 55 C for 30 secondsand 72 C for 30 seconds; and 1 cycle of 72 C for 5 minutes. The reactedsamples were stored by freezing at −20 C until being analyzed.

5 μl each of the reaction mixtures of the ICAN and the PCR was subjectedto electrophoresis on 3.0% agarose gel. The results are shown in FIG.24. FIG. 24 shows the results of the detection of a viroid according tothe ICAN using Pfu RNase H and the PCR. Lane 1: 100 bp DNA laddermarker; lane 2: negative control; lane 3: 10000-fold dilution of thecDNA; lane 4: 1000-fold dilution of the cDNA; lane 5: 100-fold dilutionof the cDNA; lane 6: 10-fold dilution of the cDNA; and lane 7: theoriginal solution of the cDNA.

As shown in FIG. 24, the amplification products of interest wereobserved using the 100-fold dilution of the cDNA for both of the ICANand the PCR.

Example 41

Detection of K-ras gene was examined.

(1) Detection from Genomic DNA

Primers c-Ki-ras-1 and c-Ki-ras-2 represented by SEQ ID NOS:215 and 216were constructed based on the nucleotide sequence of human c-Ki-ras.

10 μl of a mixture containing 60 pmol each of the primers, 2 μl of a0.25% aqueous solution of propylenediamine and 1 ng to 100 ng of humangenomic DNA (Clontech) as a template was prepared. The mixtures wereheated at 98 C for 2 minutes and then 53 C in Thermal Cycler Personal toanneal the primers to the template.

After annealing, 40 μl of a reaction mixture containing 0.625 mM each ofdNTPs, 40 mM HEPES-potassium hydroxide buffer (pH 7.8), 125 mM potassiumacetate, 5 mM magnesium acetate, 0.0125% bovine serum albumin, 1.25%dimethyl sulfoxide, 30 U of E. coli RNase H and 5.5 U of BcaBEST DNApolymerase was added to the mixture to make the final volume to 50 μl.The reaction mixtures were incubated at 53 C for 1 hour. After reaction,5 μl each of the reaction mixtures was subjected to electrophoresis on3.0% agarose gel.

On the other hand, a PCR was carried out as a control. Primersc-Ki-ras-3 and c-Ki-ras-4 represented by SEQ ID NOS:217 and 218 wereused as primers. 50 μl of a solution containing 60 pmol each of theprimers, 0.1 ng to 100 ng of the template, 5 μl of 10×Ex Taq buffer,1.25 U of TaKaRa Ex Taq polymerase and 0.2 mM each of dNTPs wasprepared. The mixtures were subjected to reactions using Thermal CyclerPersonal as follows: 30 or 35 cycles of 94 C for 30 seconds, 55 C for 30seconds and 72 C for 30 seconds. After reaction, 5 μl each of thereaction mixtures was subjected to electrophoresis on 3.0% agarose gel.The results are shown in FIG. 25. FIG. 25 shows the results of thedetection of c-Ki-ras gene from human genomic DNA according to the ICANand the PCR. For the ICAN, lane 1: molecular weight marker; lane 2: 100ng of the template; lane 3: 10 ng of the template; lane 4: 1 ng of thetemplate; and lane 5: no template. For the PCR, lane 1: 100 ng of thetemplate; lane 2: 10 ng of the template; lane 3: 1 ng of the template;and lane 4: no template.

As shown in FIG. 25, the amplification products of interest wereobserved using 1 ng of the template for the ICAN and 10 ng of thetemplate for the PCR of 30 cycles. The comparative results for theamounts of the amplification products obtained by the ICAN and the PCRusing 1 ng to 100 ng of the template are shown in FIG. 30. As shown inFIG. 30, it was confirmed that the amount of the amplification productobtained by the ICAN was greater than that by the PCR.

(2) Detection from Blood Sample

Genomic DNAs were prepared using Dr. GenTLE™ (for Whole Blood) (TakaraShuzo) from 100 μl each of blood samples collected from a healthyindividual using sodium citrate or heparin as an anticoagulating agent.Detection of c-Ki-ras gene by the ICAN was carried out as described in(1) above using the prepared DNA corresponding to 0.04 to 5 μl of theblood. Furthermore, detection by PCR using the DNA from 0.04 to 5 μl ofthe blood sample was carried out as described in (1) above in order tocompare the sensitivities for detecting a DNA by the ICAN and the PCR.The results are shown in FIG. 26. FIG. 26 shows the results of thedetection of c-Ki-ras gene from blood samples according to the ICAN andthe PCR. For the ICAN, lane 1: molecular weight marker; lane 2: 5 μl ofcitrated blood; lane 3: 1 μl of citrated blood; lane 4: 0.2 μl ofcitrated blood; lane 5: 0.04 μl of citrated blood; lane 6: 5 μl ofheparinized blood; lane 7: 1 μl of heparinized blood; lane 8: 0.2 μl ofheparinized blood; and lane 9: 0.04 μl of heparinized blood. For thePCR, lane 1: molecular weight marker; lane 2: 5 μl of citrated blood, 30cycles; lane 3: 1 μl of citrated blood, 30 cycles; lane 4: 0.2 μl ofcitrated blood, 30 cycles; lane 5: 0.04 μl of citrated blood, 30 cycles;lane 6: 5 μl of citrated blood, 35 cycles; lane 7: 1 μl of citratedblood, 35 cycles; lane 8: 0.2 μl of citrated blood, 35 cycles; lane 9:0.04 μl of citrated blood, 35 cycles; lane 10: 5 μl of heparinizedblood, 30 cycles; lane 11: 1 μl of heparinized blood, 30 cycles; lane12: 0.2 μl of heparinized blood, 30 cycles; lane 13: 0.04 μl ofheparinized blood, 30 cycles; lane 14: 5 μl of heparinized blood, 35cycles; lane 15: 1 μl of heparinized blood, 35 cycles; lane 16: 0.2 μlof heparinized blood, 35 cycles; and lane 17: 0.04 μl of heparinizedblood, 35 cycles.

As shown in FIG. 26, the amplification products of interest wereobserved using the genomic DNA corresponding to 0.2 μl of either of theblood samples for the ICAN, and the genomic DNA corresponding to 0.2 μlof either of the blood samples (citrated or heparinized) for the PCR of30 cycles, respectively.

Example 42

Detection of Escherichia coli O-157 vero toxin 2 (VT-2) gene using BcaRNase HIII was examined. Enterohemorrhagic Escherichia coli O-157 wascultured in mEC medium containing novobiocin at 42 C for 18 hours, andthen heated at 95 C for 10 minutes. Dilutions corresponding to 0, 1, 10,10² or 10³ cells in sterile water were prepared and used as templates.Primers VT-2 IF4 and VT-2 IR3 having nucleotide sequences represented bySEQ ID NOS:219 and 220 were synthesized as primers for detection. Theexpected size of the amplification product obtained using the primerpair was about 146 bp. The reaction was carried out as follows. Briefly,10 μl of a mixture containing 50 pmol each of the primers,propylenediamine at a final concentration of 0.01% and one of the hotwater-extracts was prepared. The mixtures were heated at 98 C for 2minutes and incubated at 55 C for 1 minute in Thermal Cycler Personal,and then placed on ice. At final concentrations, 34 mM Tricine buffer(pH 8.7), 10 mM potassium chloride, 10 mM ammonium sulfate, 1% dimethylsulfoxide, 0.01% bovine serum albumin, 4 mM magnesium acetate, 500 μMeach of dNTPs, 32 U of Bca RNase HIII as prepared in Referential Example3(5) and 5.5 U of BcaBEST DNA polymerase were added to the mixture tomake the final volume to 50 μl. The reaction mixtures were placed in athermal cycler which had been set at 55 C and reacted at the temperaturefor 60 minutes. After reaction, 3 μl each of the reaction mixtures wassubjected to electrophoresis on 4% NuSieve 3:1 agarose. The results areshown in FIG. 27. FIG. 27 shows the results of the detection ofEscherichia coli O-157 vero toxin II (VT2) gene using Bca RNase HIII.Lane M: molecular weight marker (100 bp ladder); lane N: sterile wateras template; lane 1: the template corresponding to 1 cell; lane 2: thetemplate corresponding to 10 cells; lane 3: the template correspondingto 10² cells; and lane 4: the template corresponding to 10³ cells.

As shown in FIG. 27, VT2 gene was detected using the hot water-extractcorresponding to 1 cell according to the ICAN. These results wereequivalent to those of the detection reactions according to the ICANusing E. coli RNase H and the PCR as described in Example 22. Thus, itwas confirmed that the ICAN using a heat-resistant RNase H, Bca RNaseHIII, was also effective in detecting a virus, a bacterium and the like.

Example 43

Detection of Staphylococcus aureus enterotoxin A gene was examined.Primers SEA-1 and SEA-2 represented by SEQ ID NOS:225 and 226 weresynthesized based on the nucleotide sequence of the enterotoxin A generegion of Staphylococcus aureus. 50 μl of a reaction mixture containing1 μl of a solution containing 115 pg or 1.15 ng of a genomic DNA fromStaphylococcus aureus (ATCC accession no. 13565) or 1 μl of water for anegative control, 50 pmol each of the primers, 0.5 mM each of dNTPs, 32mM HEPES-potassium hydroxide buffer (pH 7.8), 100 mM potassium acetate,4.0 mM magnesium acetate, 0.01% bovine serum albumin, 1.0% dimethylsulfoxide, 0.0156 pg of Pfu RNase H and 1 U of BcaBEST DNA polymerasewas incubated at 58 C for 1 hour in a thermal cycler. After reaction, 5μl each of the reaction mixtures was analyzed by electrophoresis on 3.0%agarose gel. The results are shown in FIG. 29. FIG. 29 shows the resultsof electrophoresis for the detection of Staphylococcus aureusenterotoxin A gene. Lane 1: molecular weight marker (100 bp ladder);lane 2: negative control (sterile water); lane 3: 115 pg of thetemplate; and lane 4: 1.15 ng of the template.

As shown in FIG. 29, the amplification product of interest was observedusing about 1.15 ng of the template.

Example 44

Detection of hepatitis C virus (HCV) was examined. Primers HCV-F3 andHCV-R1 having nucleotide sequences represented by SEQ ID NOS:227 and 228were synthesized based on the nucleotide sequence of HCV. A template DNAwas prepared as follows. Briefly, 4 μl of a mixture containing RNAprepared as described in Example 34 from 100 μl of a serum from ahealthy individual or a patient with HCV, 10 mM tris-hydrochloridebuffer (pH 8.3), 5 mM MgCl₂, 1 mM each of dNTPs, 10 pmol of Random 6mersprimer and 10 U of Reverse Transcriptase XL (Takara Shuzo) was incubatedat 30 C for 10 minutes and at 42 C for 1 hour and then heated at 99 Cfor 5 minutes for inactivating the enzyme using a thermal cycler (GeneAmp PCR System 9600, Applied Biosystems) to prepare a cDNA.

An ICAN was carried out at 55 C for 1 hour as described in Example 26except that 1 μl of the cDNA reaction mixture and 100 pmol each of theprimers HCV-F3 and HCV-R1 were used. After reaction, 2.5 μl each of thereaction mixtures was subjected to electrophoresis on 3.0% agarose gel.The results are shown in FIG. 31. FIG. 31 shows the results ofelectrophoresis for the detection of hepatitis C virus. Lane 1:molecular weight marker (100 bp); lane 2: the template prepared from aserum from a healthy individual; and lanes 3 to 6: the templatesprepared from sera from patients infected with HCV.

As shown in FIG. 31, it was confirmed that HCV could be specificallydetected from the serum samples from patients infected with HCV.

Example 45

The amplification method of the present invention was examined.

(1) A PCR was carried out using the pUC19-150 plasmid DNA as prepared inExample 15(2) as a template and primers MCS-F and MCS-R represented bySEQ ID NOS:124 and 125. A PCR-amplified fragment of 534 bp was obtainedby purifying the reaction mixture using Microcon-100 (Millipore). Areaction mixture containing 15 ng of the PCR fragment and 30 pmol of aprimer MR1 having a nucleotide sequence represented by SEQ ID NO:229labeled with [γ-³²P]ATP by phosphorylation at the 5′-terminus andsterile distilled water to 5 μl and a reaction mixture furthercontaining 30 pmol of a primer MR2 having a nucleotide sequencerepresented by SEQ ID NO:230 were prepared. The reaction mixtures wereheat-denatured at 98 C for 2 minutes and then cooled to 55 C. 20 μl of areaction mixture (42.5 mM Tricine buffer (pH 8.7), 12.5 mM potassiumchloride, 12.5 mM ammonium sulfate, 0.0125% BSA, 1.25% DMSO, 5 mMmagnesium acetate, 0.625 mM each of dNTPs) containing 1 U of BcaBEST DNApolymerase was added to the reaction mixture. The resulting mixture wasreacted at 55 C for 15 minutes. After reaction, 2.5 μl of a reactiontermination solution (95% formamide, 20 mM EDTA, 0.05% Bromophenol Blue,0.5% xylene cyanol) was added to 5 μl of the reaction mixture. Themixture was heat-denatured at 94 C for 3 minutes. 1.6 μl each of thereaction mixtures was subjected to electrophoresis on 6% polyacrylamidegel containing 8 M urea and the signals were read using BAS2000 (Fujix)to detect products extended from the primer MR1. The results are shownin FIG. 32A. The sequence ladder in FIG. 32A was prepared by sequencingM13 mp18 single strand DNA (Takara Shuzo) using the primer MF2 labeledwith [γ-³²P]ATP by phosphorylation and used for the determination of thelength of the extension product. Lane 1: a combination of the primersMF2 and MR1; and lane 2: MR1.

As shown in FIG. 32A, a band of 448 bp extended from the primer MR1 tothe end of the template was detected when the extension reaction wascarried out by adding only the primer MR1 to the template. On the otherhand, a band of 373 bp bounded by the primers MR1 and MF2 was detectedin addition to the above-mentioned band by further adding the primerMF2. Thus, it was confirmed that the extension from the MR1 primer usingthe PCR-amplified fragment as a template by the action of BcaBEST DNApolymerase was switched due to template switching to the extension usinga strand extended from the primer MF2 as a template. Furthermore,template switching was observed when Klenow DNA polymerase was used as amesophilic DNA polymerase having a strand displacement activity undersimilar conditions. On the other hand, template switching was notobserved when TaKaRa Taq DNA polymerase (Takara Shuzo) or PyroBEST DNApolymerase (Takara Shuzo) without a strand displacement activity wasused.

(2) The template switching reaction was examined using a template DNAstrand with a primer being annealed thereto. DNA fragments to which theprimers MF2 and MR1 could be annealed were prepared as follows. PCRswere carried out using the plasmid pUC19 as a template and primers MCSFand RV (Takara Shuzo) or primers M4 (Takara Shuzo) and MCSR. Thereaction mixtures were purified using Microcon-100 to obtainPCR-amplified fragments MSCF-RV (236 bp) and M4-MCSR (271 bp). Theregion bounded by the primers M4 and RV was commonly present in the twoPCR-amplified fragments.

Next, a template-primer (1) in which template DNA strands with primersbeing annealed thereto were not annealed each other, and atemplate-primer (2) in which template DNA strands with primers beingannealed thereto were annealed each other were prepared as follows.

(1) A reaction mixture containing 30 ng of the fragment MCSF-RV, 40 pmolof the primer MF2 labeled with [γ-³²P]ATP by phosphorylation at the5′-terminus, propylenediamine at a final concentration of 0.01% andsterile distilled water to 5 μl, and a mixture containing 30 ng of thefragment M4-MCSR, 40 pmol of the primer MR1, propylenediamine at a finalconcentration of 0.01% and sterile distilled water to 5 μl wereseparately heat-denatured at 98 C for 2 minutes and then cooled to 55 C.2.5 μl each of the reaction mixtures were mixed together to prepare atemplate-primer.

(2) A reaction mixture containing 15 ng of the fragment MCSF-RV, 15 ngof the fragment M4-MCSR, 20 pmol of the primer MF2 labeled with[γ-³²P]ATP by phosphorylation at the 5′-terminus, 20 pmol of the primerMR1, propylenediamine at a final concentration of 0.01% and steriledistilled water to 5 μl was heat-denatured at 98 C for 2 minutes andthen cooled to 55 C to prepare a template-primer.

20 μl of a reaction mixture (42.5 mM Tricine buffer (pH 8.7), 12.5 mMpotassium chloride, 12.5 mM ammonium sulfate, 0.0125% BSA, 1.25% DMSO, 5mM magnesium acetate, 0.625 mM each of dNTPs) containing 1 U of BcaBESTDNA polymerase was added to 5 μl of the template-primer reactionmixture. The resulting mixture was reacted at 55 C for 15 minutes. Afterreaction, 2.5 μl of a reaction termination solution (95% formamide, 20mM EDTA, 0.05% Bromophenol Blue, 0.5% xylene cyanol) was added to 5 μlof the reaction mixture. The mixture was heat-denatured at 94 C for 3minutes. 1.6 μl each of the reaction mixtures was subjected toelectrophoresis on 6% polyacrylamide gel containing 8 M urea and thesignals were read using BAS2000 (Fujix) to detect products extended fromthe primer MF2. The results are shown in FIG. 32B. The sequence ladderin FIG. 32B was prepared by sequencing M13 mp18 single strand DNA usingthe primer MR1 labeled with [γ-³²P]ATP by phosphorylation and used forthe determination of the length of the extension product. Lane 1:template DNA strands not being annealed each other; and lane 2: templateDNA strands being annealed each other.

As shown in FIG. 32B, only a band of 161 bp extended from the primer MF2to the end of the template was detected for the template-primer in whichtemplate DNA strands with primers being annealed thereto were notannealed each other. On the other hand, a band of 223 bp bounded by theprimers MF2 and MR1 in addition to the above-mentioned band was detectedfor the template-primer in which template DNA strands with primers beingannealed thereto were annealed each other. Thus, it was confirmed that atemplate switching reaction took place if template DNA strands withprimers being annealed thereto were annealed each other.

Example 46

(1) Detection of Mycobacterium tuberculosis was examined using the RNaseH derived from Archaeoglobus fulgidus (Afu) described in ReferentialExample 7. Primers MTIS2F (SEQ ID NO:244) and MTIS2R (SEQ ID NO:245)were synthesized on the basis of the nucleotide sequence of theMycobacterium tuberculosis genome registered in GenBank under accessionnumber AL123456. The length of the region bordered by the primer pairincluding the primer portions is 103 bp. Mycobacterium tuberculosisgenomic DNA as a template was extracted from dried BCG vaccine (NipponBCG Seizo) according to a conventional method. Solutions containing 100pg, 10 pg, 1 pg, 100 fg, 10 fg or 1 fg of the genomic DNA per μl ofsterile water were prepared. Reactions were carried out as follows.Briefly, at final concentrations, 32 mM HEPES-potassium hydroxide buffer(pH 7.8), 100 mM potassium acetate, 1% DMSO, 0.01% BSA, 4 mM magnesiumacetate, 500 μM each of dNTPs, 50 pmol each of the primers MTIS2F andMTIS2R, 8.75 U of RNase H from Afu, 8 U of BcaBEST DNA polymerase and 1μl of one of the templates were mixed and the final volume was made to50 μl with sterile water. The reaction mixtures were placed in ThermalCycler Personal which had been set at 60 C and incubated for 60 minutes.After reaction, 3 μl each of the reaction mixtures was subjected toelectrophoresis on 3.0% agarose gel.

On the other hand, a PCR was carried out as a control. Primers MTISPCR-F and MTIS PCR-R as described in Rinsho Byori (the Japanese Journalof Clinical Pathology), 43(9):941-947 (1995) were used as primers. Anamplification product of 276 bp is obtained using the primer pair. Areaction mixture of 50 μl was prepared using 10 pmol each of the primersaccording to the instruction manual attached to Ex Taq DNA polymerase(Takara Shuzo). The reaction mixture was placed in Thermal Cycler andsubjected to a reaction of 40 cycles each cycle consisting of 94 C for30 seconds, 50 C for 30 seconds and 72 C for 30 seconds. After reaction,3 μl each of the reaction mixtures was subjected to electrophoresis on3.0% agarose gel.

As a result, the amplification products of interest were observed ineither case using 100 fg of the template.

(2) Detection of Chlamydia trachomatis was examined using RNase H fromAfu or RNase H from Pyrococcus horikoshii (Pho). Primers CT2F (SEQ IDNO:246) and CT2R (SEQ ID NO:247) were synthesized on the basis of thenucleotide sequence of the Chlamydia trachomatis plasmid registered inGenBank under accession number X06707. The length of the region borderedby the primer pair including the primer portions is 109 bp. A samples asa template DNA was prepared by subjecting a clinical specimen obtainedfrom a patient agreed with the informed concent to phenol-chloroformtreatment and ethanol precipitation, and collecting a DNA. Reactionswere carried out as follows. Briefly, at final concentrations, 32 mMHEPES-potassium hydroxide buffer (pH 7.8), 100 mM potassium acetate, 1%DMSO, 0.01% BSA, 4 mM magnesium acetate, 500 μM each of dNTPs, 50 pmoleach of the primers CT2F and CT2R, 46.4 U of RNase H from Pho or 8.75 Uof RNase H from Afu, 8 U of BcaBEST DNA polymerase and 1 μl of thesample were mixed and the final volume was made to 50 μl with sterilewater. The reaction mixtures were placed in Thermal Cycler Personalwhich had been set at 55 C and incubated for 60 minutes. After reaction,3 μl each of the reaction mixtures was subjected to electrophoresis on3.0% agarose gel. As a result, the amplification products of interestwas observed. These results demonstrate that Chlamydia trachomatis canbe detected using RNase H from Afu or Pho in the method of the presentinvention.

(3) Furthermore, detection using magnetic beads in a commerciallyavailable detection instrument was examined. Briefly, an amplificationreaction was carried out as described in (1) above using the primerMTIS2R used in (1) above labeled at its 5′-terminus with biotin as aprimer and 100 ng of the Mycobacterium tuberculosis genomic DNA as atemplate. The resulting amplified fragment was diluted 30-fold, 300-foldor 3000-fold and subjected to detection using streptoavidin-coatedmagnetic beads (Pierce) in an automated detection instrument Lumipuls(Fujirebio). Magnetic beads having streptoavidin capable of binding 100pmol of biotin being immobilized was reacted with the biotinylatedamplified fragment for 5 minutes on the first layer of a cuvette. 0.1 NNaOH was then added thereto. Hybridization with a FITC-labeled probeMTISBF was carried out for 5 minutes. After washing, a POD-labeledanti-FITC antibody was added thereto. After reacting for 5 minutes andwashing, a luminescent substrate was added thereto. As a result, it wasshown that detection can be carried out semi-quantitatively in a shorttime (20 minutes) using magnetic beads in a conventional automateddetection instrument. The detection was carried out by measuring theluminescence level by photocounting. The results are shown in Table 15.TABLE 15 Mycobacterium tuberculosis amplicon Photocounting S/N ratio ×303.55 × 10⁷ 29.6 ×300 1.21 × 10⁷ 10.0 ×3000 0.21 × 10⁷ 1.75 0 0.12 × 10⁷—

The results in Table 15 demonstrate that detection can be carried outwith sensitivity equivalent to that of the conventional plateluminescence method.

(4) A hybrid chromatography method was examined as a method fordetecting the above-mentioned amplified fragment. Streptoavidin (NacalaiTesque) was immobilized onto a nitrocellulose membrane. Awater-absorptive pad was connected therewith to construct a hybridchromatography strip. This strip was used to detect the amplifiedfragment used in (3) above according to a hybrid chromatography method.Detection was carried out by color development using 1-step TMB-Blotting(Pierce). Specifically, a reaction mixture containing the amplifiedfragment was developed on the nitrocellulose membrane. Then, 0.1 N NaOHsolution, FITC-labeled probe, washing solution and color developmentsolution were developed in this order. As a result, a blue band wasdetected for the amplified fragment derived from a Mycobacteriumtuberculosis-positive sample. It was demonstrated that this method isuseful as a rapid gene examination method because, using this method,results are obtained with naked eyes in 5 to 10 minutes by the method ofthe present invention.

Example 47

(1) Southern Hybridization Analysis of Amplified Products ContainingLaddered Bands

In some cases, plural high molecular weight laddered bands other thanthree bands of interest may be observed in the amplification method ofthe present invention. The laddered bands were examined.Enterohemorrhagic E. coli O-157 was selected as a target. A templateDNA, chimeric primers and reaction conditions for ICAN were as describedin Example 22. After reaction, 5 μl of the reaction mixture wassubjected to electrophoresis on 3% NuSieve 3:1 agarose gel. The resultsare shown in FIG. 37. In FIG. 37, the lanes represent results for thefollowing: lane M: 100 bp DNA ladder marker; lane 1: negative control;lane 2: the heated extract corresponding to 1 cell; lane 3: the heatedextract corresponding to 10 cells; lane 4: the heated extractcorresponding to 10² cells; lane 5: the heated extract corresponding to10³ cells; lane 6: the heated extract corresponding to 10⁴ cells; andlane 7: the heated extract corresponding to 10⁵ cells. Ladder bands wereobserved as shown in FIG. 37.

(2) Analysis of Laddered Amplified Fragments

The nucleotide sequences of the laddered bands obtained in (1) abovewere analyzed. Briefly, 50 μl of the reaction mixture prepared in (1)was subjected to electrophoresis on 3% agarose gel. Afterelectrophoresis, the laddered bands were excised from the gel. Theamplified DNA fragments were then recovered from the gel using EASYTRAPVer.2 (Takara Shuzo). The recovered amplified fragments were blunt-endedusing DNA Blunting kit (Takara Shuzo).

The blunt-ended DNA fragments were ligated with a vector pGEM-3Z(Promega) digested with a restriction enzyme HincII (Takara Shuzo) usingDNA ligation Kit (Takara Shuzo). The ligation mixtures were used totransform competent cells of JM109 (Takara Shuzo). After transformatin,the cells were cultured on LB agar plate containing 0.1 mM ampicillin, 1mM IPTG and 0.02% X-gal at 37 C overnight.

After cultivation, several white colonies were selected from the plates.Colony PCRs were carried out using primers M13-M4 and M13-RV (both fromTakara Shuzo) to determine the presence of an insert. Colonies havinginserts were cultured with shaking in LB liquid medium containing 0.1 mMampicillin at 37 C overnight. After cultivation, plasmids were purifiedfrom cells using QIAGEN plasmid mini Kit (Qiagen). The sequences offragments cloned at the HincII sites of the plasmids were analyzed inboth directions using primers M13-M4 and M13-RV according to aconventional method.

As a result, it was demonstrated that the laddered fragment obtained bythe method of the present invention has a structure in which the regionto be amplified is repeated. Furthermore, it was confirmed that therepetition is in the same direction from 5′ to 3′.

(3) The laddered amplified fragments other than three amplifiedfragments of interest formed in the method of the present invention fordetecting Mycobacterium tuberculosis, HCV or Chlamydia trachomatis wereexamined. HCV was detected under conditions as described in Example 44.Mycobacterium tuberculosis or Chlamydia trachomatis was detected underconditions as described in Example 46. The resulting laddered amplifiedfragments were subcloned and sequenced as described in (2) above. As aresult, it was demonstrated that the laddered fragment obtained by themethod of the present invention has a structure in which the region tobe amplified is repeated. Furthermore, it was confirmed that therepetition is in the same direction from 5′ to 3′.

Example 48

(1) The detection method of the present invention for Mycobacteriumtuberculosis was examined. First, primers K-F-1033(60) (SEQ ID NO:248)and K-F-1133(62) (SEQ ID NO:249) were synthesized for amplifying aregion with relatively low GC content in the Mycobacterium tuberculosisgenome. Serial dilutions containing 100 fg to 10 pg of the Mycobacteriumtuberculosis genomic DNA as described in Example 46(1) were used astemplates. The reaction was carried our as follows. Briefly, at finalconcentrations, 32 mM HEPES-potassium hydroxide buffer (pH 7.8), 100 mMpotassium acetate, 1% DMSO, 0.01% BSA, 4 mM magnesium acetate, 500 μMeach of dNTPs, 50 pmol each of the primers K-F-1033(60) andK-F-1133(62), 9.375 U of RNase HII from Pfu or 4.375 U of RNase H fromAfu, 2.75 U of BcaBEST DNA polymerase and 1 μl of one of the templateswere mixed and the final volume was made to 25 μl with sterile water.The reaction mixtures were placed in Thermal Cycler Personal which hadbeen set at 62 C and incubated for 60 minutes. After reaction, 3 μl eachof the reaction mixtures was subjected to electrophoresis on 3.0%agarose gel. As a result, it was demonstrated that the amplificationproducts could be detected using 100 fg to 10 pg of the genomic DNA as atemplate and either RNase H.

(2) The method as described in (1) above was examined using primershaving higher Tm values. First, primers K-F-1033(68) (SEQ ID NO:250) andK-F-1133(68) (SEQ ID NO:251) were synthesized. The amplificationreaction was carried out under the same conditions as those described in(1) above except that the reaction temperature was 63 C. After reaction,3 μl each of the reaction mixtures was subjected to electrophoresis on3.0% agarose gel. The results are shown in FIG. 38. FIG. 38 illustratesresults of electrophoresis of Mycobacterium tuberculosis genomeamplified using RNase H from Pfu or RNase H from Afu. Lanes 1 to 4represent results obtained using RNase HII from Pfu and the followingamount of template DNA: lane 1: 10 pg; lane 2: 1 pg; lane 3: 100 fg; andlane 4: negative control. Lanes 5 to 8 represent results obtained usingRNase HII from Afu and the following amount of template DNA: lane 5: 10pg; lane 6: 1 pg; lane 7: 100 fg; and lane 8: negative control. Lane Mrepresents 100 bp DNA ladder marker.

As shown in FIG. 38, it was demonstrated that the amplification fragmentof interest could be detected using 100 fg of the template DNA andeither RNase H. It was shown that more amplification product is obtainedwhen RNase H from Afu is used. In addition, it was shown that morestable detection sensitivity is accomplished when RNase H from Afu isused.

(3) Amplification using a combination of the primers K-F-1033(68) andK-F-1133(68) and a plasmid containing a region to be amplified as atemplate was examined. First, primers F26 (SEQ ID NO:252) and R1310 (SEQID NO:253) were synthesized in order to prepare a plasmid containing aregion to be amplified. A PCR was carried out using these primers andBCG vaccine strain. The resulting amplification product was thenintroduced into pT7-Blue-T vector (Takara Shuzo) to prepare the plasmid.The composition of the reaction mixture was las described in (2) aboveexcept that 4 U of Bca DNA polymerase was used. As a result, it wasconfirmed that detection can be carried out using 1 fg of the template.

(4) The detection sensitivity accomplished when a combination of primersMTIS2F and MTIS2R, which results in the formation of laddered amplifiedfragments containing the three amplified fragments of interest, was usedwas compared with that accomplished when a combination of primersK-F-1033(68) and K-F-1133(68), which results in the three amplifiedfragments of interest, was used. Mycobacterium tuberculosis was selectedas a target. Reactions were carried out as described in Example 46(2)when the combination of the primers MTIS2F and MTIS2R was used or in (2)above when the combination of the primers K-F-1033(68) and K-F-1133(68)was used. As a result, the same detection sensitivity was observed ineither case.

Example 49

The amplification method of the present invention that does not comprisedenaturation of a genomic DNA as a template was examined.

(1) Primers pDON-AI-68-1 (SEQ ID NO:254) and pDON-AI-68-2 (SEQ IDNO:255) were synthesized in accordance with the nucleotide sequence ofthe packaging region in a plasmid pDON-AI (Takara Shuzo).

(2) A reaction mixture of a total volume of 50 μl containing 1 μl of asolution containing 10 fg or 1 pg of pDON-AI, 1 μl of a solutioncontaining 1 ng, 10 ng or 100 ng of the genomic DNA derived from NIH/3T3cells having pDON-AI being incorporated prepared in Example 36, or 1 p¹of water as a negative control, 50 pmol each of the primers as describedin (1) above, 0.5 mM each of dNTPs, 32 mM HEPES-potassium hydroxidebuffer (pH 7.8), 100 mM potassium acetate, 4 mM magnesium acetate, 0.01%BSA, 1% DMSO, 18.5 U of RNase HII from Pfu and 4 U of BcaBEST DNApolymerase was prepared. The reaction mixtures were placed in ThermalCycler Personal and incubated at 64 C for 1 hour. After reaction, 5 μleach of the reaction mixtures was subjected to electrophoresis on 3%agarose gel to observe amplification products. The results are shown inFIG. 39. In FIG. 39, the lanes represent results for the followingtemplates: lane M: 100 bp DNA ladder marker; lane 1: negative control;lane 2: 1 ng of genomic DNA having pDON-AI being incorporated; lane 3:10 ng of genomic DNA having pDON-AI being incorporated; lane 4: 100 ngof genomic DNA having pDON-AI being incorporated; lane 5: 10 fg ofpDNA-AI DNA; and lane 6: 1 pg of pDON-AI DNA.

As shown in FIG. 39, amplification of specific DNA fragments wasobserved for either pDON-AI or the genomic DNA having pDON-AI beingincorporated. Thus, it was confirmed that a DNA fragment of interest canbe amplified without denaturing the DNA as a template prior to thereaction even if a genomic DNA is used as a template.

Example 50

The fidelity (accuracy) of the present invention was compared with thatof a PCR utilizing LA technology in which TaKaRa Ex Taq polymerase(Takara Shuzo) was used. First, a plasmid as a template was prepared asfollows.

Specifically, the regions each consisting of 300 bp represented by SEQID NOS:256 to 259 were amplified by PCR from human proto-oncogene,Wnt-5a gene (GenBank accession no. L20861), ribosomal protein S5 gene(GenBank accession no. XM_(—)009371), human NADH gene (GenBank accessionno. NM_(—)000903) and human protocadherin 43 gene (GenBank accession no.AU077347). In this case, the PCRs were carried out using specificprimers each having a site for a restriction enzyme SfiI at the5′-terminus of the primer. After reaction, the amplified fragments weredigested with a restriction enzyme SfiI (Takara Shuzo). pUC19 (TakaraShuzo) was digested with restriction enzymes AflIII (NEB) and NdeI(Takara Shuzo) and subjected to agarose gel electrophoresis. A fragmentof about 2 kbp was excised from the gel and recovered using EASYTRAP(Takara Shuzo). A DNA having the sequence of SEQ ID NO:260 and a DNAcomplementary thereto were synthesized using a DNA synthesizer. TheseDNAs were heat-denatured and then annealed each other to form adouble-stranded DNA. This double-stranded DNA has cohesive ends forrestriction enzymes AflIII and NdeI at its termini. The double-strandedsynthetic DNA was inserted into the fragment obtained by digesting pUC19with restriction enzymes AflIII and NdeI using DNA Ligation kit ver. 2(Takara Shuzo). The resulting plasmid was designated as pIC62. pIC62 hassequences to which primers ICAN2 (SEQ ID NO:261) and ICAN6 (SEQ IDNO:262) anneal as well as a site for a restriction enzyme SfiI. Theplasmid pIC62 was digested with the restriction enzyme SfiI. Theabove-mentioned PCR-amplified fragment digested with the restrictionenzyme SfiI was ligated with the plasmid using Ligation kit ver. 2(Takara Shuzo). The ligation mixture was used to transform Escherichiacoli JM109 (Takara Shuzo). The resulting transformants were cultured,plasmids having the about 300-bp DNAs being inserted were obtained, andthe sequences of the inserted DNAs were determined. The plasmids werethen used as templates in this Example.

(2) ICAN amplification products were prepared as follows. Briefly, asolution of 10 μl containing 10 ng of one of the plasmids as a template,50 pmol each of chimeric primers ICAN2 (SEQ ID NO:261) and ICAN6 (SEQ IDNO:262) and 0.01% propylenediamine was prepared. The solution wasdenatured at 98 C for 2 minutes and incubated at 60 C for 1 minute inThermal Cycler Personal, and then transferred onto ice. Next, at finalconcentrations, 20 mM HEPES-potassium hydroxide buffer (pH 7.8), 100 mMpotassium acetate, 1% DMSO, 0.01% BSA, 4 mM magnesium acetate, 500 μMeach of dNTPs, 30 U of RNase H from E. coli and 5.5 U of BcaBEST DNApolymerase were added thereto and the final volume was made to 50 μlwith sterile water. The reaction mixtures were placed in Thermal CyclerPersonal which had been set at 60 C and incubated for 60 minutes. Afterreaction, the reaction mixtures were subjected to electrophoresis on 2%SeaKem GTG agarose gel (Takara Shuzo). After electrophoresis, bands forthe amplification products of interest were recovered by excision fromthe agarose gel, recovery using SUPREC-10 (Takara Shuzo), treatment withphenol-chloroform and ethanol precipitation.

(3) PCR amplification products were prepared as follows using TaKaRa ExTaq DNA polymerase. Briefly, a reaction mixture was prepared using 10 ngof the above-mentioned plasmid as a template as well as 10 pmol each ofDNA primers ICAN2 (SEQ ID NO:263) and ICAN6 (SEQ ID NO:264) according tothe instruction manual attached to TaKaRa Ex Taq DNA polymerase (TakaraShuzo). The reaction mixture was placed in Thermal Cycler and subjectedto a reaction of 30 cycles, each cycle consisting of 94 C for 30seconds, 55 C for 30 seconds and 72 C for 30 seconds. After reaction,the reaction mixture was subjected to agarose gel electrophoresis asdescribed in (2) above. The amplification products of interest wererecovered by excision from the agarose gel, recovery using Microcon-100(Takara Shuzo), treatment with phenol-chloroform and ethanolprecipitation.

(4) The amplification products obtained in (2) and (3) above weresubcloned as follows. Briefly, the ICAN amplification products and thePCR amplification products were introduced into a vector pT7 Blue(Takara Shuzo) using Perfectly Blunt Cloning kit (Takara Shuzo)according to the instruction manual. The ligation mixture was used totransform NovaBlue Singles Competent Cell (Takara Shuzo). For eachclone, 10 colonies were selected from the resulting transformants andcultured to obtain plasmids each having about 0.4-kb DNA being inserted.The fragments inserted in the plasmids were sequenced using T7 promoterprimer (Takara Shuzo) and M3 primer (Takara Shuzo).

A total of about 16,000 bases were analyzed by sequencing as describedabove. As a result, one mutation was found in about 25,000 bases forboth of the fragments amplified according to the method of the presentinvention and the fragments amplified by PCR using TaKaRa Ex Taq DNApolymerase. Thus, it was confirmed that the fidelity (accuracy) of themethod of the present invention is equivalent to that of LA-PCR whosefidelity is high.

Example 51

(1) Preparation of Template for ICAN Reaction by PCR

A double-stranded cDNA was prepared from polyA+ RNA derived from mousebrain (OriGene) using cDNA synthesis kit (Takara Shuzo). PCR fragmentswere amplified using the double-stranded cDNA as a template andcombinations of the primers having nucleotide sequences of SEQ IDNOS:265-278. The fragments were introduced into pT7 Blue T-vector(Takara Shuzo) by TA cloning to obtain plasmid clones. A reactionmixture of total volume of 50 μl containing 1 μl (1 ng) of one theplasmid clones, 10 pmol each of primers MCS-F (SEQ ID NO:279) and MCS-R(SEQ ID NO:280), 1.25 U of Ex Taq (Takara Shuzo), 5 μl of 10×Ex Taqbuffer (Takara Shuzo) and 0.2 mM each of dNTPs was subjected to thefollowing reaction using TaKaRa PCR Thermal Cycler Personal (TakaraShuzo): 94 C for 2 minutes; 30 cycles of 94 C for 30 seconds, 55 C for30 seconds and 72 C for 1 minute. The resulting amplified DNA fragmentwas used as a template for an ICAN reaction.

(2) Amplification of DNA Fragment by ICAN Using PCR Product as Template

An ICAN reaction was carried out using Aminoallyl dUTP (Sigma) in orderto introduce an amino group into an ICAN amplification product. Theratio of amino group introduced into an ICAN amplification product wasexamined by changing the ratio of the amount of dTTP to the amount ofAminoallyl dUTP in the ICAN reaction as follows: 10:0, 9:1, 8:2, 7:3 and6:4. The reaction was carried out as follows.

First, a solution of total volume of 10 μl containing 1 μl of the PCRreaction mixture prepared in (1) above, 50 pmol each of primersMF2N3(24) (SEQ ID NO:281) and MR1N3(24) (SEQ ID NO:282) and 2 μl of0.05% aqueous solution of propylenediamine was heated at 98 C for 2minutes followed by 65 C for 30 seconds in TaKaRa PCR Thermal CyclerPersonal and rapidly cooled on ice to anneal the primers to thetemplate. A reaction mixture of total volume of 40 μl containing 0.625mM each of DATP, dCTP and dGTP, 0.625 mM of a dTTP+Aminoallyl dUTPmixture, 32 mM HEPES-potassium hydroxide buffer (pH 7.8), 5.0 mMmagnesium acetate, 0.6 U of RNase H from E. coli (Takara Shuzo) and 2.75U of BcaBEST DNA polymerase was added to the heated solution. Theresulting mixture was incubated in Thermal Cycler at 65 C for 1 hour.

50 μl of isopropanol and 5 μl of 3 M sodium acetate solution (pH 5.2)were added to the reaction mixture of 50 μl. The resulting mixture wascooled at −80 C for 20 minutes and then centrifuged to remove asupernatant. 200 μl of 70% ethanol solution was added thereto. Thesupernatant was removed by centrifugation. The precipitate wasair-dried. The resulting DNA was re-dissolve in water. OD_(260/280) wasmeasured to determined the amount of the product.

(3) Confirmation of Introduction of Aminoallyl dUTP into ICANAmplification Product

Introduction of an amino group into an ICAN product was confirmed byfluorescence-label the amino group in the ICAN product using5-carboxyfluorecein succinimidil ester (Molecular Probe). Theabove-mentioned DNA solution was diluted to make the concentration to 2μg/50 μl. 20 μl of 1 M sodium carbonate buffer (pH 9.0) was addedthereto. Then, 4 μl of a solution of FITC (Nacalai Tesque) inN,N-dimethylformamide at a concentration of 10 mM was further added. Theresulting mixture was reacted at 20 C for 16 hours. After removingexcess FITC using a commercially available spin column, 10 μl of thereaction mixture was applied onto 2.0% agarose gel and electrophoresed.After electrophoresis, fluorescent dye was detected using FM-BIO.Furthermore, the ICAN-amplified fragment was detected by staining withEtBr. As a result, it was confirmed that an amino group can beintroduced into an ICAN amplification product by conducting an ICANusing Aminoallyl dUTP. It was also confirmed that the detectionsensitivity can be further increased by using a modified nucleotidehaving a functional group and fluorescent label for an amplificationproduct in combination.

(4) The amplification method of the present invention was examined usingdeoxyUTP. Mycobacterium tuberculosis was selected as a target. First,primers MTIS2F-16 (SEQ ID NO:283) and MTIS2R-ACC (SEQ ID NO:284) weresynthesized in accordance with the nucleotide sequence of Mycobacteriumtuberculosis genome registered in GenBank under accession no. AL123456.The length of the region to be amplified using this primer pairincluding the primer portions is 98 bp. A template was prepared asfollows. Briefly, a product obtained by PCR-amplifying the Mycobacteriumtuberculosis genome using primers MTIS-PCR-F-2 (SEQ ID NO:285) andMTIS-PCR-R-2 (SEQ ID NO:286) was inserted into pT7 Blue T-Vector (TakaraShuzo) using DNA Ligation kit Ver. 2. The ligated plasmid was used totransform Escherichia coli JM109. The resulting transformants werecultured to obtain a plasmid having an about 400-bp DNA beingintroduced. A solution containing 10³ copies of the plasmid per μl wasprepared based on the concentration determined by measuring OD260.

At final concentrations, 32 mM HEPES-potassium hydroxide buffer (pH7.8), 100 mM potassium acetate, 1% DMSO, 0.01% BSA, 4 mM magnesiumacetate, 500 μM each of DATP, dCTP, dGTP and a dTTP/dUTP mixture (500/0,400/100, 300/200, 200/300, 100/400 or 0/500 μM), 50 pmol each of theprimers MTIS 2F-16 and MTIS 2R-AAC, 8.75 U of RNase H from Afu, 8 U ofBcaBEST DNA polymerase and 1 μl of the template (10³ copies) were mixedand the final volume was made to 50 μl with sterile water. The reactionmixtures were placed in Thermal Cycler Personal which had been set at 60C and incubated for 60 minutes. After reaction, 3 μl each of thereaction mixtures was subjected to electrophoresis on 3% agarose gel.

As a result, the amplification product of interest was observed for eachof the dTTP/dUTP ratios. Based on these results, it was confirmed that amodified nucleotide can be used as a substrate in the method of thepresent invention.

Example 52

Application of the method of the present invention to a one-stepamplification method was examined. HCV was selected as a target.

(1) Preparation of Transcript RNA

A transcript RNA as a template was prepared. At first, HCV RNA wasprepared from 300 μl of a serum derived from a patient with hepatitis Cvirus agreed with the informed concent using TRIzol reagent (LifeTechnologies) according to the instructions attached to the reagent andfinally dissolved in 20 μl of injectable water (Otsuka Pharmaceutical)to obtain an RNA sample. An RT-PCR was carried out using the RNA sampleabove as a template. The reaction was carried out as follows. A reactionmixture of 50 μl was prepared using 2 μl of the RNA sample and 20 pmoleach of primers SP6—HCV-F (SEQ ID NO:287) and T7-HCV-R (SEQ ID NO:288)according to a manual attached to One-Step RNA PCR kit (Takara Shuzo).The reaction mixture was placed in Thermal Cycler Personal and subjectedto the following reaction: 50 C for 15 minutes; 94 C for 2 minutes; and40 cycles of 94 C for 30 seconds, 60 C for 30 seconds and 72 C for 30seconds. After reaction, the reaction mixture was subjected toelectrophoresis on 2% SeaPlaque GTG agarose gel. A 350-bp amplificationproduct of interest was excised from the gel. The DNA was then recoveredusing EASYTRAP Ver. 2 according to the instructions attached to the kit.A transcript RNA was synthesized using the recovered DNA as a templateand Competitive RNA Transcription kit (Takara Shuzo) according to theinstructions attached to the kit. The transcript RNA was used as atemplate for examination of a one-step RT-ICAN.

(2) Examination of One-Step RT-ICAN

The concentration of the transcript RNA prepared in (1) above wasdetermined based on the OD₂₆₀ value and dilutions containing 10⁴, 10⁵,10⁶ or 10⁷ copies per μl were prepared. A reaction mixture of 50 μl wasprepared by adding thereto, at final concentrations, 32 mMHEPES-potassium hydroxide buffer (pH 7.8), 100 mM potassium acetate, 1%DMSO, 0.01% BSA, 4 mM magnesium acetate, 500 μM each of dNTPs, 50 pmoleach of the primers HCV-A S (SEQ ID NO:289) and HCV-A A (SEQ ID NO:290),30 U of RNase H from Pfu, 8 U of BcaBEST DNA polymerase, 20 U of RNaseinhibitor, 0, 1, 2.5, 3 or 5 U of AMV RTase XL (Takara Shuzo) and 1 μlof one of the dilutions containing varying copies of the transcript RNA.The reaction mixtures were placed in Thermal Cycler Personal which hadbeen set at 60 C and incubated for 60 minutes. After reaction, 2 μl eachof the reaction mixtures was subjected to electrophoresis on 3% agarosegel.

As a result, no amplification product of interest was observed usingeach of the dilutions of the template when AMV RTase was not added. Onthe other hand, the amplification product of interest was observed usingthe dilution containing 10⁷ copies (1 U of AMV RTase XL added), 10⁶copies (2.5 U of AMV RTase XL added), 10⁶ copies (3 U of AMV RTase XLadded) or 10⁶ copies (5 U of AMV RTase XL added). When the reaction wascarried out at a temperature of 57 C, the amplification product ofinterest was observed using the dilution containing 10⁵ copies and 2.5 Uof AMV RTase XL. Furthermore, when 1 U of BcaBEST DNA polymenrase and 10U of RNase H from Pfu were used, the amplification product of interestwas observed using the dilution containing 10⁶ copies even if no AMVRTase was added.

Sequence Listing Free Text

SEQ ID No:1: Synthetic DNA corresponding to a portion of humantransferrin receptor-encoding sequence used as a template

SEQ ID NO:2: Designed oligonucleotide primer to amplify a portion ofhuman transferrin receptor-encoding sequence

SEQ ID NO:3: Designed oligonucleotide primer to amplify a portion ofhuman transferrin receptor-encoding sequence

SEQ ID No:4: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotide 21is ribonucleotide—other nucleotides are deoxyribonucleotides”

SEQ ID No:5: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotide 21is ribonucleotide—other nucleotides are deoxyribonucleotides”

SEQ ID No:6: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotide 22is ribonucleotide—other nucleotides are deoxyribonucleotides”

SEQ ID No:7: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotide 22is ribonucleotide—other nucleotides are deoxyribonucleotides”

SEQ ID No:8: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:9: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:10: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 19to 20 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No: 11: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 19to 20 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:12: Designed oligonucleotide used as a probe for detecting anamplified portion of human transferrin receptor-encoding sequence

SEQ ID No: 13: Designed chimeric oligonucleotide primer designated aspUC19 upper(2)NN to amplify a portion of plasmid PUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No: 14: Designed chimeric oligonucleotide primer designated aspUC19 lower NN to amplify a portion of plasmid pUC19. “nucleotides 24 to25 are ribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID No:15: Designed chimeric oligonucleotide primer to amplify aportion of plasmid pUC19. “nucleotides 24 to 25 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:16: Designed chimeric oligonucleotide primer designated aspUC19 lower 542 to amplify a portion of plasmid PUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:17: Designed chimeric oligonucleotide primer to amplify aportion of plasmid pUC19. “nucleotides 24 to 25 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:18: Designed oligonucleotide primer designated as pUC19 upper150 to amplify a portion of plasmid pUC19. “nucleotides 23 to 25 areribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID No:19: Designed chimeric oligonucleotide primer designated aspUC19 lower NN to amplify a portion of plasmid pUC19. “nucleotides 23 to25 are ribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID No:20: Designed chimeric oligonucleotide primer designated aspUC19 upper 249 to amplify a portion of plasmid PUC19. “nucleotides 23to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No: 21: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 20to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:22: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:23: Designed chimeric oligonucleotide primer designated aspUC19 upper(2)NN to amplify a portion of plasmid pUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No: 24: Designed chimeric oligonucleotide primer designated aspUC19 upper(2)NN to amplify a portion of plasmid pUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:25: Designed chimeric oligonucleotide primer designated aspUC19 upper(2)NN to amplify a portion of plasmid pUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:26: Designed chimeric oligonucleotide primer designated aspUC19 upper(2)NN to amplify a portion of plasmid PUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:27: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:28: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:29: Designed chimeric oligonucleotide primer designated asMF2N3(24) to amplify a portion of plasmid pUC19-249 or plasmidpUC19-911. “nucleotides 22 to 24 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID No:30: Designed oligonucleotide primer designated as MR1N3(24) toamplify a portion of plasmid pUC19-249 or plasmid pUC19-911.“nucleotides 22 to 24 are ribonucleotides—other nucleotides aredeoxyribonucleotides”

SEQ ID No:31: Designed chimeric oligonucleotide primer designated aspUC19 upper 249 to amplify a portion of plasmid pUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:32: Designed oligonucleotide primer designated as pUC19 upper150 to amplify a portion of plasmid pUC19

SEQ ID No:33: Designed oligonucleotide primer designated as pUC19 upper249 to amplify a portion of plasmid pUC19

SEQ ID No:34: Designed oligonucleotide primer designated as pUC19 lowerNN to amplify a portion of plasmid pUC19

SEQ ID No:35: Designed chimeric oligonucleotide primer to amplify aportion of plasmid pUC19. “nucleotides 28 to 30 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:36: Designed chimeric oligonucleotide primer designated asMR1N3 to amplify a portion of plasmid pUC19. “nucleotides 28 to 30 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:37: Designed oligonucleotide primer to amplify a portion ofplasmid pUC19

SEQ ID No:38: Designed oligonucleotide primer designated as MR1N3 toamplify a portion of plasmid pUC19

SEQ ID No:39: Synthetic RNA used as a probe for detecting an amplifiedportion of plasmid pUC19

SEQ ID No:40: Designed chimeric oligonucleotide primer designated aspUC19 upper 150 to amplify a portion of plasmid pUC19. “nucleotides 24to 25 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:41: Designed chimeric oligonucleotide primer designated asMR1N3 to amplify a portion of plasmid pUC19. “nucleotides 28 to 30 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:42: Designed oligonucleotide primer designated as M13M4

SEQ ID No:43: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 1-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 16 to 18 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID No:44: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 1-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 15 to 17 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID No:45: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 16 to 18 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID No:46: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 16 to 18 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID No:47: Designed oligonucleotide primer designated as MCR-F toamplify a long DNA fragment

SEQ ID No:48: Designed oligonucleotide primer designated as MCR-R toamplify a long DNA fragment

SEQ ID No:49: Designed chimeric oligonucleotide primer designated asMF2N3(24) to amplify a long DNA fragment. “nucleotides 22 to 24 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:50: Designed chimeric oligonucleotide primer designated asMR1N3(24) to amplify a long DNA fragment. “nucleotides 22 to 24 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:51: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:52: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:53: Designed chimeric oligonucleotide primer to amplify aportion of bacteriophage lambda DNA. “nucleotides 16 to 17 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:54: Designed chimeric oligonucleotide primer to amplify aportion of bacteriophage lambda DNA. “nucleotides 16 to 17 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:55: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:56: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:57: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:58: Designed oligonucleotide primer designated as R1-S1 toamplify a portion of bacteriophage lambda DNA

SEQ ID No:59: Designed oligonucleotide primer designated as R1-A3 toamplify a portion of bacteriophage lambda DNA

SEQ ID No:60: Designed oligonucleotide primer designated as R2-S1 toamplify a portion of bacteriophage lambda DNA

SEQ ID No:61: Designed oligonucleotide primer designated as R2-A3 toamplify a portion of bacteriophage lambda DNA

SEQ ID No:62: Designed oligonucleotide primer designated as R3-S1 toamplify a portion of bacteriophage lambda DNA

SEQ ID No:63: Designed oligonucleotide primer designated as R3-A3 toamplify a portion of bacteriophage lambda DNA

SEQ ID No:64: Designed chimeric oligonucleotide primer designated asM13RV-2N 17mer. “nucleotides 16 to 17 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID No:65: Designed chimeric oligonucleotide primer designated asM13RV-2N 20mer. “nucleotides 19 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID No:66: Designed oligonucleotide primer to amplify a portion ofCDC2-related protein kinase PISSLRE gene

SEQ ID No:67: Designed oligonucleotide primer to amplify a portion ofCDC2-related protein kinase PISSLRE gene

SEQ ID No:68: Designed oligonucleotide primer to amplify a portion ofType II cytoskeltal 11 keratin gene

SEQ ID No:69: Designed oligonucleotide primer to amplify a portion ofType II cytoskeltal 11 keratin gene

SEQ ID No:70: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:71: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:72: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:73: Designed oligonucleotide primer to amplify a portion ofbacteriophage lambda DNA

SEQ ID No:74: Designed oligonucleotide primer designated as MF2N3(24) toamplify a portion of plasmid pUC19-249 or plasmid pUC19-911

SEQ ID No:75: Designed oligonucleotide primer designated as MR1N3(24) toamplify a portion of plasmid pUC19-249 or plasmid pUC19-911

SEQ ID No:76: Designed chimeric oligonucleotide primer designated asM13M4-3N 20mer. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID No:77: Designed chimeric oligonucleotide primer designated asM13RV-3N 20mer. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID No:78: Designed chimeric oligonucleotide primer designated asM13M4-3N 24mer. “nucleotides 22 to 24 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID No:79: Designed oligonucleotide primer designated as M13RV-3N24mer. “nucleotides 22 to 24 are ribonucleotides—other nucleotides aredeoxyribonucleotides”

SEQ ID No:80: Designed oligonucleotide primer designated as 5′ID toamplify a portion of cyclin A DNA

SEQ ID No:81: Designed oligonucleotide primer designated as 3′ID toamplify a portion of cyclin A DNA

SEQ ID No:82: Designed oligonucleotide primer designated as M13RV-2N16mer. “nucleotides 15 to 16 are ribonucleotides—other nucleotides aredeoxyribonucleotides”

SEQ ID No:83: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:84: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:85: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:86: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:87: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:88: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:89: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:90: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:91: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:92: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No: 93: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:94: Designed chimeric oligonucleotide primer to amplify aportion of human transferrin receptor-encoding sequence. “nucleotides 21to 22 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID No:95: Designed oligonucleotide primer to amplify a portion ofhuman transferrin receptor-encoding sequence

SEQ ID No:96: Designed oligonucleotide primer to amplify a portion ofhuman transferrin receptor-encoding sequence

SEQ ID NO:97: PCR primer BsuII-3 for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:98: PCR primer BsuII-6 for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:99: PCR primer RNII-S1 for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:100: PCR primer RNII-S2 for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:101: PCR primer RNII-S5 for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:102: PCR primer RNII-S6 for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:103: PCR primer RNII-Nde for cloning a gene encoding apolypeptide having a RNaseHII activity from Bacillus caldotenax.

SEQ ID NO:104: Nucleotide sequence of ORF in RNaseHII gene from Bucilluscaldotenax.

SEQ ID NO:105: Amino acid sequence of RNaseHII from Bucillus caldotenax.

SEQ ID NO:106: PCR primer BsuIII-1 for cloning a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:107: PCR primer BsuIII-3 for cloning a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:108: PCR primer BsuIII-6 for cloning a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:109: PCR primer BsuIII-8 for cloning a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:110: PCR primer RNIII-S3 for cloning a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:111: PCR primer BcaRNIII-3 for cloning a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:112: Nucleotide sequence of ORF in RNaseHIII from Bacilluscaldotenax.

SEQ ID NO:113: Amino acid sequence of RNaseHIII from Bacilluscaldotenax.

SEQ ID NO:114: PCR primer BcaRNIIINde for amplifying a gene encoding apolypeptide having a RNaseHIII activity from Bacillus caldotenax.

SEQ ID NO:115: Nucleotide sequence conserving between PH1650 and aportion of Pyrococcus furiosus genome sequence.

SEQ ID NO:116: PCR primer 1650Nde for cloning a gene encoding apolypeptide having a RNaseHII activity from Pyrococcus furiosus.

SEQ ID NO:117: PCR primer 1650Bam for cloning a gene encoding apolypeptide having a RNaseHII activity from Pyrococcus furiosus.

SEQ ID NO:118: Nucleotide sequence of ORF in RNaseHII from Pyrococcusfuriosus.

SEQ ID NO:119: Amino acid sequence of RNaseHII from Pyrococcus furiosus.

SEQ ID NO:120: PCR primer 915-F1 for cloning a gene encoding apolypeptide having a RNaseHII activity from Thermotoga maritima.

SEQ ID NO:121: PCR primer 915-F2 for cloning a gene encoding apolypeptide having a RNaseHII activity from Thermotoga maritima.

SEQ ID NO:122: PCR primer 915-R1 for cloning a gene encoding apolypeptide having a RNaseHII activity from Thermotoga maritima.

SEQ ID NO:123: PCR primer 915-R2 for cloning a gene encoding apolypeptide having a RNaseHII activity from Thermotoga maritima.

SEQ ID NO:124: Designed oligonucleotide primer designated as MCR-F toamplify a long DNA fragment

SEQ ID NO:125: Designed oligonucleotide primer designated as MCR-R toamplify a long DNA fragment

SEQ ID NO:126: Designed chimeric oligonucleotide primer designated asMF2N3(24) to amplify a long DNA fragment. “nucleotides 22 to 24 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:127: Designed chimeric oligonucleotide primer designated asMR1N3(24) to amplify a long DNA fragment. “nucleotides 22 to 24 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:128: Designed oligonucleotide primer to amplify a portion oflambda DNA. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:129: Designed chimeric oligonucleotide primer to amplify aportion of lambda DNA. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:130: Designed oligonucleotide primer to amplify a portion oflambda DNA

SEQ ID NO:131: Designed oligonucleotide primer to amplify a portion oflambda DNA

SEQ ID NO:132: Designed chimeric oligonucleotide primer to amplify aportion of Flavobacterium species DNA. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:133: esigned chimeric oligonucleotide primer to amplify aportion of Flavobacterium species DNA. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:134: Designed oligonucleotide primer to amplify a portion ofFlavobacterium species DNA.

SEQ ID NO:135: Designed oligonucleotide primer to amplify a portion ofFlavobacterium species DNA.

SEQ ID NO:136: esigned chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 19 to 21 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:137: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:138: Designed oligonucleotide primer to amplify a portion ofvero toxin 2-encoding sequence from hemorrhagic Escherichia coli O-157.

SEQ ID NO:139: Designed oligonucleotide primer to amplify a portion ofvero toxin 2-encoding sequence from hemorrhagic Escherichia coli O-157.

SEQ ID NO:140: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:141: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:142: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:143: Designed oligonucleotide primer to amplify a portion ofvero toxin 2-encoding sequence from hemorrhagic Escherichia coli O-157.

SEQ ID NO:144: Designed oligonucleotide primer to amplify a portion ofvero toxin 2-encoding sequence from hemorrhagic Escherichia coli O-157.

SEQ ID NO:145: Designed chimeric oligonucleotide primer to amplify aportion of lambda DNA. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:146: Designed oligonucleotide primer to amplify a portion ofviroid CSVd.

SEQ ID NO:147: Designed oligonucleotide primer to amplify a portion ofviroid CSVd.

SEQ ID NO:148: Designed chimeric oligonucleotide primer to amplify aportion of viroid CSVd. “nucleotides 16 to 18 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:149: Designed chimeric oligonucleotide primer to amplify aportion of viroid CSVd. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:150: Designed chimeric oligonucleotide primer to amplify aportion of Flavobacterium species DNA. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:151: Designed chimeric oligonucleotide primer to amplify aportion of Flavobacterium species DNA. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:152: Designed chimeric oligonucleotide primer to amplify aportion of Flavobacterium species DNA. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:153: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 19 to 21 are ribonucleotides-nucloetide 18 isinosine—other nucleotides are deoxyribonucleotides”

SEQ ID NO:154: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 19 to 21 are ribonucleotides-nucleotide 17 isinosine other nucleotides are deoxyribonucleotides”

SEQ ID NO:155: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 19 to 21 are ribonucleotides-nucleotide 16 isinosine—other nucleotides are deoxyribonucleotides”

SEQ ID NO:156: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides-nucleotide 17 isinosine—other nucleotides are deoxyribonucleotides”

SEQ ID NO:157: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides-nucleotide 16 isinosine—other nucleotides are deoxyribonucleotides”

SEQ ID NO:158: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides-nucleotide 15 isinosine—other nucleotides are deoxyribonucleotides”

SEQ ID NO:159: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 9 to 11 and 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:160: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 8 to 10 and 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:161: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:162: Designed oligonucleotide probe to detect a DNA fragmentamplifing a portion of vero toxin 2-encoding sequence from hemorrhagicEscherichia coli O-157.

SEQ ID NO:163: Designed chimeric oligonucleotide primer to amplify aportion of iNOS-encoding sequence from mouse. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:164: Designed chimeric oligonucleotide primer to amplify aportion of iNOS-encoding sequence from mouse. “nucleotides 17 to 19 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:165: Designed oligonucleotide primer to amplify a portion ofiNOS-encoding sequence from mouse.

SEQ ID NO:166: Designed oligonucleotide primer to amplify a portion ofiNOS-encoding sequence from mouse

SEQ ID NO:167: Designed oligonucleotide primer designated as GMO-PCR-F20mer

SEQ ID NO:168: Designed oligonucleotide primer designated as GMO-PCR-R20mer

SEQ ID NO:169: Designed chimeric oligonucleotide primer designated asGMO-S1 20mer. “nucleotides 19 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:170: Designed oligonucleotide primer designated as GMO-S220mer. “nucleotides 19 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides”

SEQ ID NO:171: Designed oligonucleotide primer designated as GMO-A120mer. “nucleotides 19 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides”

SEQ ID NO:172: Designed oligonucleotide primer designated as GMO-A2 20mer. “nucleotides 19 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides”

SEQ ID NO:173: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are (alpha-thio)ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:174: Designed chimeric oligonucleotide primer to amplify aportion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are (alpha-thio)ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:175: Designed chimeric oligonucleotide primer to amplify aportion of INOS-encoding sequence from mouse. “nucleotides 20 to 22 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:176: Designed chimeric oligonucleotide primer to amplify aportion of INOS-encoding sequence from mouse. “nucleotides 20 to 22 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:177: Designed oligonucleotide primer to amplify a portion ofINOS-encoding sequence from mouse.

SEQ ID NO:178: Designed oligonucleotide primer to amplify a portion ofINOS-encoding sequence from mouse.

SEQ ID NO:179: Designed chimeric oligonucleotide primer to amplify aportion of lambda DNA. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:180: Designed chimeric oligonucleotide primer to amplify aportion of lambda DNA. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:181: Designed chimeric oligonucleotide primer to amplify aportion of INOS-encoding sequence from mouse. “nucleotides 21 to 23 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:182: Designed chimeric oligonucleotide primer to amplify aportion of INOS-encoding sequence from mouse. “nucleotides 20 to 22 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:183: Designed chimeric oligonucleotide primer to amplify aportion of pDON-AI DNA. “nucleotides 17 to 19 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:184: Designed chimeric oligonucleotide primer to amplify aportion of pDON-AI DNA. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:185: Designed chimeric oligonucleotide primer to amplify aportion of HPV DNA. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:186: Designed chimeric oligonucleotide primer to amplify aportion of HPV DNA. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:187: Designed oligonucleotide probe to detect a DNA fragmentamplifing a portion of HPV DNA.

SEQ ID NO:188: Designed oligonucleotide primer to amplify a portion ofHCV.

SEQ ID NO:189: Designed oligonucleotide primer to amplify a portion ofHCV.

SEQ ID NO:190: Designed chimeric oligonucleotide primer to amplify aportion of HCV. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:191: Designed chimeric oligonucleotide primer to amplify aportion of HCV. “nucleotides 16 to 18 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:192: Designed oligonucleotide probe to detect a DNA fragmentamplifing portion of HCV.

SEQ ID NO:193: Designed chimeric oligonucleotide primer to amplify aportion of adenovirus. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:194: Designed chimeric oligonucleotide primer to amplify aportion of adenovirus. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:195: Designed chimeric oligonucleotide primer to amplify aportion of adenovirus. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:196: Designed oligonucleotide primer to amplify a portion ofadenovirus

SEQ ID NO:197: Designed oligonucleotide primer to amplify a portion ofadenovirus.

SEQ ID NO:198: Designed oligonucleotide primer to amplify a portion ofviroid CSVd.

SEQ ID NO:199: Designed oligonucleotide primer to amplify a portion ofviroid CSVd.

SEQ ID NO:200: Designed oligonucleotide primer to amplify a portion ofpDON-AI DNA.

SEQ ID NO:201: Designed oligonucleotide primer to amplify a portion ofpDON-AI DNA.

SEQ ID NO:202: Designed chimeric oligonucleotide primer to amplify aportion of verotoxin-1 encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:203: Designed chimeric oligonucleotide primer to amplify aportion of verotoxin-1 encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:204: Designed oligonucleotide probe to detect a DNA fragmentamplifying a portion of verotoxin-1 encoding sequence from hemorrhagicEscherichia coli O-157.

SEQ ID NO:205: Designed chimeric oligonucleotide primer to amplify aportion of botulinum toxin A encoding sequence from Clostridiumbotulinum. “nucleotides 19 to 21 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:206: Designed chimeric oligonucleotide primer to amplify aportion of botulinum toxin A encoding sequence from Clostridiumbotulinum. “nucleotides 21 to 23 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:207: Designed oligonucleotide probe to detect a DNA fragmentamplifying a portion of botulinum toxin A encoding sequence fromClostridium botulinum.

SEQ ID NO:208: Designed chimeric oligonucleotide primer to amplify aportion of viroid CSVd. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:209: Designed chimeric oligonucleotide primer to amplify aportion of viroid CSVd. “nucleotides 18 to 20 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:210: Designed oligonucleotide probe to detect a DNA fragmentamplifying a portion of viroid CSVd.

SEQ ID NO:211: Designed chimeric oligonucleotide primer to amplify aportion of viroid CSVd. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:212: Designed chimeric oligonucleotide primer to amplify aportion of viroid CSVd. “nucleotides 19 to 21 are ribonucleotides—othernucleotides are deoxyribonucleotides”

SEQ ID NO:213: Designed oligonucleotide primer to amplify a portion ofviroid CSVd.

SEQ ID NO:214: Designed oligonucleotide primer to amplify a portion ofviroid CSVd.

SEQ ID NO:215: Designed chimeric oligonucleotide primer to amplify aportion of c-ki-ras oncogene. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:216: Designed chimeric oligonucleotide primer to amplify aportion of c-ki-ras oncogene. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:217: Designed oligonucleotide primer to amplify a portion ofc-ki-ras oncogene.

SEQ ID NO:218: Designed oligonucleotide primer to amplify a portion ofc-ki-ras oncogene.

SEQ ID NO:219: Designed chimeric oligonucleotide primer to amplify aportion of verotoxin-1 encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:220: Designed chimeric oligonucleotide primer to amplify aportion of verotoxin-1 encoding sequence from hemorrhagic Escherichiacoli O-157. “nucleotides 18 to 20 are ribonucleotides—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:221: Designed oligonucleotide primer to amplify a portion ofINOS-encoding sequence from mouse.

SEQ ID NO:222: Designed oligonucleotide primer to amplify a portion ofINOS-encoding sequence from mouse.

SEQ ID NO:223: Designed oligonucleotide primer designated as pUC19 upper150 to amplify a portion of plasmid pUC19.

SEQ ID NO:224: Designed oligonucleotide primer designated as pUC19 lowerNN to amplify a portion of plasmid pUC19.

SEQ ID NO:225: Designed chimeric oligonucleotide primer designated asSEA-1 to amplify a portion of Staphylococcus aureus. “nucleotides 19 to21 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:226: Designed chimeric oligonucleotide primer designated asSEA-2 to amplify a portion of Staphylococcus aureus. “nucleotides 19 to21 are ribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:227: Designed chimeric oligonucleotide primer designated asHCV-F3 to amplify a portion of HCV. “nucleotides 17 to 19 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:228: Designed chimeric oligonucleotide primer designated asHCV-R1 to amplify a portion of HCV. “nucleotides 16 to 18 areribonucleotides—other nucleotides are deoxyribonucleotides”

SEQ ID NO:229: Designed oligonucleotide primer designated as MF2 toamplify a portion of pUC19 plasmid DNA.

SEQ ID NO:230: Designed oligonucleotide primer designated as MR1 toamplify a portion of pUC19 plasmid DNA.

SEQ ID NO:231: Designed oligonucleotide primer to amplify a portion ofadenovirus.

SEQ ID NO:232: Nucleotide sequence of ORF in RNaseHII gene fromThermotoga maritima.

SEQ ID NO:233: Amino acid sequence of RNaseHII from Thermotoga maritima.

SEQ ID NO:234: Nucleotide sequence of PH1650 from Pyrococcus horikoshii.

SEQ ID NO:235: PCR primer PhoNde for cloning a gene encoding apolypeptide having a RNaseHII activity from Pyrococcus horikoshii

SEQ ID NO:236: PCR primer PhoBam for cloning a gene encoding apolypeptide having a RNaseHII activity from Pyrococcus horikoshii

SEQ ID NO:237: Nucleotide sequence of ORF in RNaseHII gene fromPyrococcus horikoshii.

SEQ ID NO:238: Amino acid sequence of RNaseHII from Pyrococcushorikoshii.

SEQ ID NO:239: Nucleotide sequence of AF0621 from Archaeoglobusfulgidus.

SEQ ID NO:240: PCR primer AfuNde for cloning a gene encoding apolypeptide having a RNaseHII activity from Archaeoglobus fulgidus

SEQ ID NO:241: PCR primer AfuBam for cloning a gene encoding apolypeptide having a RNaseHII activity from Archaeoglobus fulgidus

SEQ ID NO:242: Nucleotide sequence of ORF in RNaseHII gene fromArchaeoglobus fulgidus.

SEQ ID NO:243: Amino acid sequence of RNaseHII from Archaeoglobusfulgidus.

SEQ ID NO:244: Designed chimeric oligonucleotide primer designated asMTIS2F to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 16 to 18 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:245: Designed chimeric oligonucleotide primer designated asMTIS2R to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 19 to 21 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:246: Designed chimeric oligonucleotide primer designated asCT2F to amplify a portion of Chlamydia trachomatis cryptic plasmid DNA.“nucleotides 18 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:247: Designed chimeric oligonucleotide primer designated asCT2R to amplify a portion of Chlamydia trachomatis cryptic plasmid DNA.“nucleotides 16 to 18 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:248: Designed chimeric oligonucleotide primer designated asK-F-1033(60) to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 17 to 19 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:249: Designed chimeric oligonucleotide primer designated asK-R-1133(62) to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 18 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:250: Designed chimeric oligonucleotide primer designated asK-F-1033(68) to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 20 to 22 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:251: Designed chimeric oligonucleotide primer designated asK-R-1133(68) to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 20 to 22 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:252: Designed oligonucleotide primer designated as F26 toamplify a portion of Mycobacterium tuberculosis DNA.

SEQ ID NO:253: Designed oligonucleotide primer designated as R1310 toamplify a portion of Mycobacterium tuberculosis DNA.

SEQ ID NO:254: Designed chimeric oligonucleotide primer designated aspDON-AI-68-1 to amplify a portion of pDON-AI. “nucleotides 20 to 22 areribonucleotides—other nucleotides are deoxyribonucleotides.”.

SEQ ID NO:255: Designed chimeric oligonucleotide primer designated aspDON-AI-68-2 to amplify a portion of pDON-AI. “nucleotides 21 to 23 areribonucleotides—other nucleotides are deoxyribonucleotides.”

SEQ ID NO:256: Nucleotide sequence of Homo sapiens proto-oncogene Wnt-5a

SEQ ID NO:257: Nucleotide sequence of Homo sapiens ribosomal protein S5

SEQ ID NO:258: Nucleotide sequence of Homo sapiens diaphorase

SEQ ID NO:259: Nucleotide sequence of Human protocadherin

SEQ ID NO:260: Designed oligonucleotide for making of pIC62.

SEQ ID NO:261: Designed chimeric oligonucleotide primer designated asICAN2. “nucleotides 19 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”

SEQ ID NO:262: Designed chimeric oligonucleotide primer designated asICAN6. “nucleotides 19 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”.

SEQ ID NO:263: Designed oligonucleotide primer designated as ICAN2 DNA.

SEQ ID NO:264: Designed oligonucleotide primer designated as ICAN6 DNA.

SEQ ID NO:265: Designed oligonucleotide primer to amplify a portion ofribosomal protein S18-encoding sequence from mouse.

SEQ ID NO:266: Designed oligonucleotide primer to amplify a portion ofribosomal protein S18-encoding sequence from mouse.

SEQ ID NO:267: Designed oligonucleotide primer to amplify a portion oftransferrin receptor (TFR)-encoding sequence from mouse.

SEQ ID NO:268: Designed oligonucleotide primer to amplify a portion oftransferrin receptor (TFR)-encoding sequence from mouse.

SEQ ID NO:269: Designed oligonucleotide primer to amplify a portion ofstromal cell derived factor 4 (Sdf4)-encoding sequence from mouse.

SEQ ID NO:270: Designed oligonucleotide primer to amplify a portion ofstromal cell derived factor 4 (Sdf4)-encoding sequence from mouse.

SEQ ID NO:271: Designed oligonucleotide primer to amplify a portion ofcytoplasmic beta-actin encoding sequence from mouse.

SEQ ID NO:272: Designed oligonucleotide primer to amplify a portion ofcytoplasmic beta-actin encoding sequence from mouse.

SEQ ID NO:273: Designed oligonucleotide primer to amplify a portion ofornithine decarboxylase-encoding sequence from mouse.

SEQ ID NO:274: Designed oligonucleotide primer to amplify a portion ofornithine decarboxylase-encoding sequence from mouse.

SEQ ID NO:275: Designed oligonucleotide primer to amplify a portion ofhypoxanthine guanine phosphoribosyl transferase (HPRT)-encoding sequencefrom mouse.

SEQ ID NO:276: Designed oligonucleotide primer to amplify a portion ofhypoxanthine guanine phosphoribosyl transferase (HPRT)-encoding sequencefrom mouse.

SEQ ID NO:277: Designed oligonucleotide primer to amplify a portion oftyrosine 3-monooxygenase encoding sequence from mouse.

SEQ ID NO:278: Designed oligonucleotide primer to amplify a portion oftyrosine 3-monooxygenase encoding sequence from mouse.

SEQ ID NO:279: Designed oligonucleotide primer designated as MCS-F.

SEQ ID NO:280: Designed oligonucleotide primer designated as MCS-R

SEQ ID NO:281: Designed chimeric oligonucleotide primer designated asMF2N3(24). “nucleotides 22 to 24 are ribonucleoitdes—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:282: Designed chimeric oligonucleotide primer designated asMR1N3(24). “nucleotides 22 to 24 are ribonucleoitdes—other nucleotidesare deoxyribonucleotides”

SEQ ID NO:283: Designed chimeric oligonucleotide primer designated asMTIS2F-16 to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 14 to 16 are ribonucleotides—other nucleotides aredeoxyribonucleotides.

SEQ ID NO:284: Designed chimeric oligonucleotide primer designated asMTIS2R-ACC to amplify a portion of Mycobacterium tuberculosis DNA.“nucleotides 18 to 20 are ribonucleotides—other nucleotides aredeoxyribonucleotides.”

SEQ ID NO:285: Designed oligonucleotide primer designated asMTIS-PCR-F-2 to amplify a portion of Mycobacterium tuberculosis DNA

SEQ ID NO:286: Designed oligonucleotide primer designated asMTIS-PCR-R-2 to amplify a portion of Mycobacterium tuberculosis DNA

SEQ ID NO:287: Designed oligonucleotide primer designated as SP6—HCV-Fto amplify a portion of HCV

SEQ ID NO:288: Designed oligonucleotide primer designated as SP6—HCV-Rto amplify a portion of HCV

SEQ ID NO:289: Designed chimeric oligonucleotide primer designated asHCV-A S to amplify a portion of HCV. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides.”

SEQ ID NO:290: Designed chimeric oligonucleotide primer designated asHCV-A A to amplify a portion of HCV. “nucleotides 18 to 20 areribonucleotides—other nucleotides are deoxyribonucleotides.

1. A method for amplifying a nucleic acid, characterized in that themethod comprises: (a) preparing a reaction mixture by mixing a nucleicacid as a template, a deoxyribonucleotide triphosphate, a DNA polymerasehaving a strand displacement activity, at least one primer and anendonuclease that cleaves an extended strand generated from the primer,wherein the primer is a chimeric oligonucleotide primer that issubstantially complementary to the nucleotide sequence of the nucleicacid as the template and contains a ribonucleotide as well as at leastone selected from the group consisting of a deoxyribonucleotide and anucleotide analog, the ribonucleotide being positioned at the3′-terminus or on the 3′-terminal side of the primer; and (b) incubatingthe reaction mixture for a sufficient time to generate a reactionproduct under conditions where specific annealing of the primer to thenucleic acid as the template, an extended strand synthesis reaction anda strand displacement reaction by the DNA polymerase, and a reaction ofcleaving the extended strand by the endonuclease take place.
 2. Themethod according to claim 1, wherein the reaction mixture is incubatedisothermally.
 3. The method according to claim 1, wherein the reactionmixture further contains another chimeric oligonucleotide primer havinga sequence substantially homologous to the nucleotide sequence of thenucleic acid as the template.
 4. The method according to claim 1,wherein the DNA polymerase is selected from the group consisting ofKlenow fragment of DNA polymerase I from Escherichia coli, Bst DNApolymerase lacking 5′→3′ exonuclease from Bacillus stearothermophilusand Bca DNA polymerase lacking 5′→3′ exonuclease from Bacilluscaldotenax.
 5. The method according to claim 1, wherein the endonucleaseis an endoribonuclease.
 6. The method according to claim 5, wherein theendoribonuclease is RNase H.
 7. The method according to claim 6, whereinthe RNase H is selected from the group consisting of an RNase H fromEscherichia coli, an RNase H from a bacterium of genus Thermotoga, anRNase H from a bacterium of genus Thermus, an RNase H from a bacteriumof genus Pyrococcus, an RNase H from a bacterium of genus Archaeoglobusand an RNase H from a bacterium of genus Bacillus.
 8. The methodaccording to claim 1, wherein the DNA polymerase having a stranddisplacement activity is Bca DNA polymerase lacking 5′→3′ exonucleasefrom Bacillus caldotenax and the RNase H as an endonuclease is selectedfrom the group consisting of an RNase H from Escherichia coli, an RNaseH from a bacterium of genus Pyrococcus and an RNase H from a bacteriumof genus Archaeoglobus.
 9. The method according to claim 8, wherein theRNase H is type I RNase H from Escherichia coli, or type II RNase H froma bacterium of genus Pyrococcus or a bacterium of genus Archaeoglobus.10. The method according to claim 1, wherein a DNA polymerase having anendonuclease activity is used.
 11. The method according to claim 10,wherein the DNA polymerase is Bca DNA polymerase lacking 5′→3′exonuclease from Bacillus caldotenax and the Bca DNA polymerase is usedin the presence of a substance that allows the endonuclease activity ofthe Bca DNA polymerase to express.
 12. The method according to claim 11,wherein the substance that allows the endonuclease activity of the DNApolymerase to express is a manganese ion.
 13. The method according toclaim 1, wherein the amplification reaction is conducted in the presenceof a substance that inhibits the reverse transcription activity of theDNA polymerase.
 14. The method according to claim 13, wherein thesubstance that inhibits the reverse transcription activity of the DNApolymerase is phosphonoformic acid.
 15. The method according to claim 1,wherein the chimeric oligonucleotide primer contains two or moresuccessive ribonucleotide residues.
 16. The method according to claim 1,wherein the chimeric oligonucleotide primer contains one or moremodified ribonucleotide.
 17. The method according to claim 16, whereinthe chimeric oligonucleotide primer contains an (α-S) ribonucleotide inwhich the oxygen atom bound to the phosphorous atom at the α-position ofthe ribonucleotide is replaced by a sulfur atom.
 18. The methodaccording to claim 1, wherein a chimeric oligonucleotide primerrepresented by general formula below is used:5′-dNa-Nb-dNc-3′  General formula wherein a is an integer of 11 or more;b is an integer of 1 or more; c is 0 or an integer of 1 or more; dN isan deoxyribonucleotide and/or nucleotide analog; N is an unmodifiedribonucleotide and/or modified ribonucleotide, wherein some of dNs indNa may be replaced by Ns, and the nucleotide at the 3′-terminus may bemodified such that extension from the 3′-terminus by the action of theDNA polymerase does not take place.
 19. The method according to claim18, wherein c is
 0. 20. The method according to claim 18, wherein thenucleotide analog is deoxyriboinosine nucleotide or deoxyribouracilnucleotide, and the modified ribonucleotide is (α-S) ribonucleotide. 21.The method according to claim 18, wherein the nucleic acid amplificationreaction is conducted at a temperature suitable for the chimericoligonucleotide primer as defined in claim
 18. 22. The method accordingto claim 1, wherein the chimeric oligonucleotide primer is at least onechimeric oligonucleotide primer selected from the group consisting of:a) a chimeric oligonucleotide primer for detecting enterohemorrhagicEscherichia coli having a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 43 to 46, 136, 137, 140-142, 153-161, 173,174, 202, 203, 219 and 220; b) a chimeric oligonucleotide primer fordetecting a viroid having a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 148, 149, 208, 209, 211, 212; c) a chimericoligonucleotide primer for detecting Clostridium botulinum having anucleotide sequence represented by SEQ ID NO: 205 or 206; d) a chimericoligonucleotide primer for detecting papilloma virus having a nucleotidesequence represented by SEQ ID NO: 185 or 186; e) a chimericoligonucleotide primer for detecting hepatitis C virus having anucleotide sequence selected from the group consisting of SEQ ID NOS:190, 191, 227, 228, 289 and 290; f) a chimeric oligonucleotide primerfor detecting Staphylococcus aureus having a nucleotide sequencerepresented by SEQ ID NO: 225 or 226; g) a chimeric oligonucleotideprimer for detecting Mycobacterium tuberculosis having a nucleotidesequence selected from the group consisting of SEQ ID NOS: 244, 245, 248to 251, 283, 284; and h) a chimeric oligonucleotide primer for detectingChlamydia having a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 246 and
 247. 23. The method according to claim1, which is conducted in a buffer that contains a buffering componentselected from the group consisting of Tricine, a phosphate, tris, Bicineand HEPES.
 24. The method according claim 1, wherein the nucleic acid asthe template is a single-stranded DNA or a double-stranded DNA.
 25. Themethod according to claim 24, which is conducted after converting adouble-stranded DNA as the template into single-stranded DNAs.
 26. Themethod according to claim 24, wherein the nucleic acid as the templateis a cDNA obtained from an RNA by a reverse transcription reaction. 27.The method according to claim 26, which is conducted after synthesizinga cDNA by a reverse transcription reaction using an RNA as a template.28. The method according to claim 27, wherein a primer selected from thegroup consisting of an oligo-dT primer, a random primer and a specificprimer is used as a primer for the reverse transcription reaction. 29.The method according to claim 27, wherein a chimeric oligonucleotideprimer is used as a primer for the reverse transcription reaction. 30.The method according to claim 27, wherein a DNA polymerase having areverse transcriptase activity is used as a reverse transcriptase. 31.The method according to claim 27, wherein the reverse transcriptionreaction and the nucleic acid amplification reaction are conducted usingone DNA polymerase having a reverse transcriptase activity and a stranddisplacement activity.
 32. The method according to claim 31, wherein theDNA polymerase is Bst DNA polymerase lacking 5′→3′ exonuclease fromBacillus stearothermophilus or Bca DNA polymerase lacking 5′→3′exonuclease from Bacillus caldotenax.
 33. The method according to claim26, wherein the RNA as the template in the reverse transcriptionreaction is an RNA amplified by an additional nucleic acid amplificationreaction.
 34. The method according to claim 33, which is conducted aftersynthesizing an amplified RNA fragment by an additional nucleic acidamplification reaction using an RNA as a template.
 35. The methodaccording to claim 33, wherein the additional nucleic acid amplificationreaction is selected from the group consisting of thetranscription-based amplification system (TAS) method, theself-sustained sequence replication (3SR) method, the nucleic acidsequence-based amplification (NASBA) method, the transcription-mediatedamplification (TMA) method and the Qβ replicase method.
 36. The methodaccording to claim 24, wherein the nucleic acid as the template is a DNAobtained by an additional nucleic acid amplification reaction.
 37. Themethod according to claim 36, which is conducted after synthesizing anamplified DNA fragment by an additional nucleic acid amplificationreaction using an DNA as a template.
 38. The method according to claim36, wherein the additional nucleic acid amplification reaction isselected from the group consisting of the polymerase chain reaction(PCR) method, the ligase chain reaction (LCR) method and the stranddisplacement amplification (SDA) method.
 39. The method according toclaim 33, wherein a random primer or a degenerate primer is used for theadditional nucleic acid amplification reaction.
 40. The method accordingto claim 39, wherein the random primer or the degenerate primer is aprimer having a random sequence or a degenerate sequence at least at the3′-terminus or on the 3′-terminal side.
 41. The method according toclaim 36, wherein a random primer or a degenerate primer is used for theadditional nucleic acid amplification reaction.
 42. The method accordingto claim 41, wherein the random primer or the degenerate primer is aprimer having a random sequence or a degenerate sequence at least at the3′-terminus or on the 3′-terminal side.
 43. The method according toclaim 1, wherein the length of the region of the nucleic acid to beamplified is 200 bp or shorter.
 44. The method according to claim 1,which comprises annealing the nucleic acid as the template to thechimeric oligonucleotide primer that is substantially complementary tothe nucleotide sequence of the nucleic acid in an annealing solutioncontaining a substance that enhances the annealing of the nucleic acidto the primer.
 45. The method according to claim 44, wherein theannealing solution contains spermidine and/or propylenediamine.
 46. Themethod according to claim 44, wherein the annealing is conducted byincubating the annealing solution containing the nucleic acid as thetemplate and the chimeric oligonucleotide primer that is substantiallycomplementary to the nucleotide sequence of the nucleic acid at 90° C.or above and then cooling the solution to a temperature at which theamplification reaction is conducted or below.
 47. The method accordingto claim 1, wherein the nucleic acid amplification reaction is conductedin the presence of a deoxyribonucleotide triphosphate analog.
 48. Themethod according to claim 47, wherein the deoxyribonucleotidetriphosphate analog is deoxyuridine triphosphate or a derivativethereof.
 49. The method according to claim 1, wherein a templateswitching reaction is effected.
 50. The method according to claim 1,which further comprises a step of duplicating a DNA or an RNA containinga sequence to be amplified to prepare a nucleic acid as a template priorto step (a), wherein the duplicated nucleic acid is used in step (a) asa nucleic acid as a template.
 51. A method for producing a nucleic acidin large quantities, characterized in that the method comprises: (a)amplifying a nucleic acid by the method defined by claim 1; and (b)collecting the nucleic acid amplified in step (a).
 52. A method fordetecting a target nucleic acid in a sample, characterized in that themethod comprises: (a) amplifying a target nucleic acid by the methoddefined by claim 1; and (b) detecting the target nucleic acid amplifiedin step (a).
 53. The method according to claim 52, which comprisesdetecting the amplified nucleic acid using a probe for detection. 54.The method according to claim 53, wherein the probe for detection is aprobe that has been labeled with a labeling substance.
 55. The methodaccording to claim 54, wherein the probe is an RNA probe labeled withtwo or more fluorescent substances positioned at a distance that resultsin a quenching state.
 56. The method according to claim 53, wherein theprobe is a probe which hybridizes to a region amplified using at leastone chimeric oligonucleotide primer selected from the group consistingof: a) a chimeric oligonucleotide primer for detecting enterohemorrhagicEscherichia coli having a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 43 to 46, 136, 137, 140-142, 153-161, 173,174, 202, 203, 219 and 220; b) a chimeric oligonucleotide primer fordetecting a viroid having a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 148, 149, 208, 209, 211 and 212; c) a chimericoligonucleotide primer for detecting Clostridium botulinum having anucleotide sequence represented by SEQ ID NO: 205 or 206; d) a chimericoligonucleotide primer for detecting papilloma virus having a nucleotidesequence represented by SEQ ID NO: 185 or 186; e) a chimericoligonucleotide primer for detecting hepatitis C virus having anucleotide sequence selected from the group consisting of SEQ ID NOS:190, 191, 227, 228, 289 and 290; f) a chimeric oligonucleotide primerfor detecting Staphylococcus aureus having a nucleotide sequencerepresented by SEQ ID NO: 225 or 226; g) a chimeric oligonucleotideprimer for detecting Mycobacterium tuberculosis having a nucleotidesequence selected from the group consisting of SEQ ID NOS: 244, 245, 248to 251, 283, 284; and h) a chimeric oligonucleotide primer for detectingChlamydia having a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 246 or
 247. 57. A method for determining anucleotide sequence of a nucleic acid, characterized in that the methodcomprises: (a) amplifying a nucleic acid by the method defined by claim1; and (b) determining the nucleotide sequence of the nucleic acidamplified in step (a).
 58. A method for preparing a single-strandednucleic acid, the method comprising generating a single-stranded nucleicacid using the method defined by claim
 1. 59. The method according toclaim 58, wherein at least two primers at different concentrations areused.
 60. A method for amplifying a nucleotide sequence, characterizedin that the method comprises: (a) treating a nucleic acid as a templatewith at least one primer that is substantially complementary to thenucleotide sequence of the nucleic acid and a DNA polymerase tosynthesize a primer-extended strand that is complementary to thetemplate, wherein the primer is a chimeric oligonucleotide primercontaining a deoxyribonucleotide and a ribonucleotide, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (b) cleaving the primer-extended strand of adouble-stranded nucleic acid obtained in step (a) with an endonucleaseat a site that contains the ribonucleotide; and (c) extending anucleotide sequence that is complementary to the template using a DNApolymerase having a strand displacement activity from the 3′-terminus ofthe primer portion of the double-stranded nucleic acid in which theprimer-extended strand is cleaved obtained in step (b) to effect astrand displacement.
 61. A method for amplifying a nucleotide sequenceusing at least two primers, characterized in that the method comprises:(a) treating a nucleic acid as a template with at least one primer thatis substantially complementary to the nucleotide sequence of the nucleicacid and a DNA polymerase to synthesize a primer-extended strand that iscomplementary to the template, wherein the primer is a chimericoligonucleotide primer containing a deoxyribonucleotide and aribonucleotide, the ribonucleotide being positioned at the 3′-terminusor on the 3′-terminal side of the primer; (b) cleaving theprimer-extended strand of a double-stranded nucleic acid obtained instep (a) with an endonuclease at a site that contains theribonucleotide; (c) extending a nucleotide sequence that iscomplementary to the template using a DNA polymerase having a stranddisplacement activity from the 3′-terminus of the primer portion of thedouble-stranded nucleic acid in which the primer-extended strand iscleaved obtained in step (b) to effect a strand displacement, wherein adouble-stranded nucleic acid containing a regenerated primer-extendedstrand is reused in step (b); (d) treating a released displaced strandobtained in step (c) as a template with at least one primer that isdifferent from that used in step (a) and a DNA polymerase to synthesizea primer-extended strand that is complementary to the displaced strand,wherein the primer that is different from that used in step (a) is achimeric oligonucleotide primer that is substantially complementary tothe nucleotide sequence of the displaced strand and contains adeoxyribonucleotide and a ribonucleotide, the ribonucleotide beingpositioned at the 3′-terminus or on the 3′-terminal side of the primer;(e) cleaving the primer-extended strand of a double-stranded nucleicacid obtained in step (d) with an endonuclease at a site that containsthe ribonucleotide; and (f) extending a nucleotide sequence that iscomplementary to the template using a DNA polymerase having a stranddisplacement activity from the 3′-terminus of the primer portion of thedouble-stranded nucleic acid in which the primer-extended strand iscleaved obtained in step (e) to effect a strand displacement, wherein adouble-stranded nucleic acid containing a regenerated primer-extendedstrand is reused in step (e).
 62. A method for amplifying a nucleicacid, characterized in that the method comprises: (a) treating a nucleicacid as a template with at least one primer that is substantiallycomplementary to the nucleotide sequence of the nucleic acid and a DNApolymerase to synthesize a primer-extended strand that is complementaryto the template and synthesize a double-stranded nucleic acid, whereinthe primer is a chimeric oligonucleotide primer containing aribonucleotide as well as at least one selected from the groupconsisting of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (b) extending a nucleic acid that is complementaryto the double-stranded nucleic acid as a template obtained in theprevious step using a DNA polymerase having a strand displacementactivity in the presence of an RNase H to effect a strand displacementand synthesize a displaced strand and a double-stranded nucleic acid;and (c) reusing in step (b) the double-stranded nucleic acid obtained instep (b) as a template.
 63. The method according to claim 62, whereinthe DNA polymerase in step (a) is different from the DNA polymerasehaving a strand displacement activity in step (b).
 64. A method foramplifying a nucleic acid using at least two primers, characterized inthat the method comprises: (a) treating a nucleic acid as a templatewith at least one primer that is substantially complementary to thenucleotide sequence of the nucleic acid and a DNA polymerase tosynthesize a primer-extended strand that is complementary to thetemplate, wherein the primer is a chimeric oligonucleotide primercontaining a ribonucleotide as well as at least one selected from thegroup consisting of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (b) extending a nucleic acid that is complementaryto the double-stranded nucleic acid as a template obtained in theprevious step using a DNA polymerase having a strand displacementactivity in the presence of an RNase H to effect a strand displacementand synthesize a displaced strand and a double-stranded nucleic acid;(c) reusing in step (b) the double-stranded nucleic acid obtained instep (b) as a template; (d) treating a displaced strand obtained in step(b) as a template with at least one primer that is different from thatused in step (a) and a DNA polymerase to synthesize a primer-extendedstrand that is complementary to the displaced strand, wherein the primerthat is different from that used in step (a) is a chimericoligonucleotide primer that is substantially complementary to thenucleotide sequence of the displaced strand and contains aribonucleotide as well as at least one selected from the groupconsisting of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (e) extending a nucleic acid that is complementaryto the double-stranded nucleic acid as a template obtained in theprevious step using a DNA polymerase having a strand displacementactivity in the presence of an RNase H to effect a strand displacementand synthesize a displaced strand and a double-stranded nucleic acid;and (f) reusing in step (e) the double-stranded nucleic acid obtained instep (e) as a template.
 65. The method according to claim 64, whereinthe DNA polymerase in steps (a) and (d) is different from the DNApolymerase having a strand displacement activity in steps (b) and (f).66. A method for amplifying a nucleic acid, characterized in that themethod comprises: (a) treating a double-stranded nucleic acid as atemplate with two primers that are substantially complementary to thenucleotide sequences of the respective strands of the double-strandednucleic acid and a DNA polymerase having a strand displacement activityto synthesize primer-extended strands that are complementary to thetemplate and obtain a double-stranded nucleic acid consisting of thesynthesized primer-extended strands being annealed each other, whereineach primer is a chimeric oligonucleotide primer containing aribonucleotide as well as at least one selected from the groupconsisting of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (b) cleaving the sites that contain theribonucleotide of the double-stranded nucleic acid consisting of theprimer-extended strands obtained in step (a) with the endonuclease; and(c) extending nucleic acids that are complementary to the template usinga DNA polymerase having a strand displacement activity from the3′-termini of the respective primer portions of the double-strandednucleic acid in which the primer-extended strands are cleaved obtainedin step (b) to effect strand displacements and obtain a double-strandednucleic acid consisting of the template and the primer-extended strand.67. A method for amplifying a nucleic acid, characterized in that themethod comprises: (a) treating a double-stranded nucleic acid as atemplate with two primers that are substantially complementary to thenucleotide sequences of the respective strands of the double-strandednucleic acid and a DNA polymerase having a strand displacement activityto synthesize primer-extended strands that are complementary to thetemplate and obtain a double-stranded nucleic acid consisting of thesynthesized primer-extended strands being annealed each other, whereineach primer is a chimeric oligonucleotide primer containing aribonucleotide as well as at least one selected from the groupconsisting of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (b) cleaving the sites that contain theribonucleotide of the double-stranded nucleic acid consisting of theprimer-extended strands obtained in step (a) with the endonuclease; and(c) extending nucleic acids that are complementary to the template usinga DNA polymerase having a strand displacement activity from the3′-termini of the respective primer portions of the double-strandednucleic acid in which the primer-extended strands are cleaved obtainedin step (b) to effect strand displacements and obtain a double-strandednucleic acid consisting of the primer-extended strands being annealedeach other.
 68. A method for amplifying a nucleic acid, characterized inthat the method comprises: (a) treating a double-stranded nucleic acidas a template with two primers that are substantially complementary tothe nucleotide sequences of the respective strands of thedouble-stranded nucleic acid and a DNA polymerase having a stranddisplacement activity to synthesize primer-extended strands that arecomplementary to the template and obtain a double-stranded nucleic acidconsisting of the synthesized primer-extended strands being annealedeach other, wherein each primer is a chimeric oligonucleotide primercontaining a ribonucleotide as well as at least one selected from thegroup consisting of a deoxyribonucleotide and a nucleotide analog, theribonucleotide being positioned at the 3′-terminus or on the 3′-terminalside of the primer; (b) cleaving the sites that contain theribonucleotide of the double-stranded nucleic acid consisting of theprimer-extended strands obtained in step (a) with the endonuclease; (c)extending nucleic acids that are complementary to the template using aDNA polymerase having a strand displacement activity from the 3′-terminiof the respective primer portions of the double-stranded nucleic acid inwhich the primer-extended strands are cleaved obtained in step (b) toeffect strand displacements and obtain a double-stranded nucleic acidconsisting of the primer-extended strands being annealed each other anda double-stranded nucleic acid consisting of the templates beingannealed each other to which the two primers in step (a) are annealed;(d) extending nucleic acids that are complementary to the template usinga DNA polymerase having a strand displacement activity from the3′-termini of the respective primer portions of the double-strandednucleic acid to which the two primers are annealed obtained in step (c)to effect strand displacements and obtain a double-stranded nucleic acidconsisting of the primer-extended strands being annealed each other anda double-stranded nucleic acid consisting of the templates beingannealed each other to which the two primers in step (a) are annealed;and (e) reusing in step (d) the double-stranded nucleic acid to whichthe two primers are annealed obtained in step (d).
 69. A method foramplifying a nucleic acid, characterized in that the method comprises:(a) treating a double-stranded nucleic acid as a template with twoprimers that are substantially complementary to the nucleotide sequencesof the respective strands of the double-stranded nucleic acid and a DNApolymerase having a strand displacement activity to synthesizeprimer-extended strands that are complementary to the template andobtain a double-stranded nucleic acid consisting of the synthesizedprimer-extended strands being annealed each other, wherein each primeris a chimeric oligonucleotide primer containing a ribonucleotide as wellas at least one selected from the group consisting of adeoxyribonucleotide and a nucleotide analog, the ribonucleotide beingpositioned at the 3′-terminus or on the 3′-terminal side of the primer;(b) cleaving the sites that contain the ribonucleotide of thedouble-stranded nucleic acid consisting of the primer-extended strandsobtained in step (a) with the endonuclease; (c) extending nucleic acidsthat are complementary to the template using a DNA polymerase having astrand displacement activity from the 3′-termini of the respectiveprimer portions of the double-stranded nucleic acid in which theprimer-extended strands are cleaved obtained in step (b) to effectstrand displacements and obtain a double-stranded nucleic acidconsisting of the primer-extended strands being annealed each other anda double-stranded nucleic acid consisting of the templates beingannealed each other to which the two primers in step (a) are annealed;(d) extending nucleic acids that are complementary to the template usinga DNA polymerase having a strand displacement activity from the3′-termini of the respective primer portions of the double-strandednucleic acid to which the two primers are annealed obtained in step (c)to effect strand displacements and obtain a double-stranded nucleic acidconsisting of the template and the primer-extended strand; (e) cleavingthe sites that contain the ribonucleotide of the double-stranded nucleicacid consisting of the template and the primer-extended strand obtainedin step (d) with the endonuclease; and (f) extending a nucleic acid thatis complementary to the template using a DNA polymerase having a stranddisplacement activity from the 3′-terminus of the primer portion of thedouble-stranded nucleic acid in which the primer-extended strand iscleaved obtained in step (e) to synthesize a displaced strand.
 70. Themethod according to claim 60, wherein step (b) and step (c) aresequentially repeated.
 71. The method according to claim 60, wherein theDNA polymerase in step (a) is different from the DNA polymerase having astrand displacement activity in step (c).
 72. The method according toclaim 61, wherein one DNA polymerase having a strand displacementactivity is used.